ALTERED EXPRESSION OF Y CHROMOSOME-LINKED ANTIGENS IN HYPOIMMUNOGENIC CELLS

EXPRESSION MODIFIEE D'ANTIGENES LIES AU CHROMOSOME Y DANS DES CELLULES HYPO-IMMUNOGENES

English Abstract

Disclosed herein are engineered cells and/or hypoimmunogenic cells including engineered cells and/or hypoimmunogenic stem cells, engineered cells and/or hypoimmunogenic cells differentiated therefrom, and engineered cells and/or hypoimmunogenic CAR-T cells (primary or differentiated from engineered and/or hypoimmunogenic stem cells) and related methods of their use and generation comprising reduced expression of one or more Y chromosome genes and reduced expression of MHC I and/or MHC II human leukocyte antigen molecules and overexpression of CD47. Provided herein are cells further exhibiting reduced expression of T-cell receptors.

French Abstract

Des cellules modifiées et/ou des cellules hypo-immunogènes comprenant des cellules modifiées et/ou des cellules souches hypo-immunogènes, des cellules modifiées et/ou des cellules hypo-immunogènes différenciées à partir de celles-ci et des cellules modifiées et/ou des cellules CAR-T hypo-immunogènes (primaires ou différenciées à partir de cellules souches génétiquement modifiées et/ou hypo-immunogènes) sont divulguées, ainsi que des procédés associés de leur utilisation et de leur génération comprenant une expression réduite d'un ou de plusieurs gènes de chromosome Y, une expression réduite de molécules d'antigène de leucocyte humain CMH I et/ou CMH II et la surexpression de CD47. La divulgation concerne des cellules présentant en outre une expression réduite des récepteurs des lymphocytes T.

Claims

WHAT IS CLAIMED IS:
1. An engineered cell comprising reduced expression of one or more Y
chromosome
genes and major histocompatibility complex (1VIHC) class I and/or class II
human leukocyte
antigen molecules relative to an unaltered or unmodified wild-type or control
cell, and a first
exogenous polynucleotide encoding CD47, wherein the engineered cell is
propagated from a
primary T cell or a progeny thereof, or is derived from an induced pluripotent
stem cell (iPSC) or
a progeny thereof.
2. A hypoimmunogenic T cell comprising reduced expression of one or more Y
chromosome genes and1VITIC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the hypoimmunogenic T cell is propagated from a primary
T cell or a
progeny thereof, or is derived from an iPSC or a progeny thereof.
3. A non-activated T cell comprising reduced expression of one or more Y
chromosome genes and IVIEIC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the non-activated T cell is propagated from a primary T
cell or a
progeny thereof, or is derived from an iPSC or a progeny thereof.
4. A pancreatic islet cell comprising reduced expression of one or more Y
chromosome genes and WIC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the pancreatic islet cell is derived from an iPSC or a
progeny thereof.
5. A cardiac muscle cell comprising reduced expression of one or more Y
chromosome genes and1VILIC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the cardiac muscle cell is derived from an iPSC or a
progeny thereof.
6. A glial progenitor cell comprising reduced expression of one or more Y
chromosome genes and 1VIEIC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the cardiac muscle cell is derived from an iPSC or a
progeny thereof.
289

7. A NK cell comprising reduced expression of one or more Y chromosome
genes
and MI-IC class I and/or class II human leukocyte antigen molecules relative
to an unaltered or
unmodified wild-type or control cell, and a first exogenous polynucleotide
encoding CD47,
wherein the cardiac muscle cell is derived from an iPSC or a progeny thereof
8. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-7, wherein
the Y chromosome gene is a Y chromosome linked antigen or a minor
histocompatibility antigen
associated with the Y chromosome.
9. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim 8,
wherein the one or
more Y chromosome linked antigens are Protocadherin-11 Y-linked and/or
Neuroligin-4 Y-
linked.
10. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-9, wherein
the cell has reduced expression of Protocadherin-11 Y-linked.
11. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-10, wherein
the cell has reduced expression of Neuroligin-4 Y-linked.
12. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-11, wherein
the cell has reduced expression of Protocadherin-11 Y-linked and reduced
expression of
Neuroligin-4 Y-linked.
13. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-12, wherein
the cell is genetically engineered to have reduced expression of Protocadherin-
11 Y-linked
and/or Neuroligin-4 Y-linked.
14. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-13, wherein
the cell does not express Protocadherin-11 Y-linked
290

15. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-14, wherein
the cell does not express Neuroligin-4 Y-linked.
16. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-15, wherein
the cell does not express Protocadherin-11 Y-linked and does not express
Neuroligin-4 Y-linked.
17. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-16, wherein
the cell is genetically engineered to not express Protocadherin-11 Y-linked
and/or Neuroligin-4
Y-linked.
18. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-17, wherein
reduced expression of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked
is caused by a
knock out of the PCDH11Y and/or NLGN4Y gene, respectively.
19. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-18, wherein
the cell is derived from a human cell or an animal cell.
20. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
19, wherein the human
cell or animal cell is from a donor subject that does not have a Y chromosome.
21. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, gli al progenitor cell, or NK cell of claim
19, wherein the human
cell or animal cell is from a donor subject that has a Y chromosome, and
wherein the cell is
genetically engineered to have reduced expression of Protocadherin-11 Y-linked
and/or
Neuroligin-4 Y-linked.
22. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
21, wherein the cell is
genetically engineered to not express Protocadherin-11 Y-linked.
291

23. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
21, wherein the cell is
genetically engineered to not express Neuroligin-4 Y-linked.
24. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
21, wherein the cell is
genetically engineered to not express Protocadherin-11 Y-linked and to not
express Neuroligin-4
Y-linked.
25. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-24, wherein
the cell is propagated or derived from a pool of cells that are isolated from
one or more donor
subjects different from the patient, wherein the one or more donor subjects
optionally comprise
one or more subjects that have a Y chromosome; one or more subjects that do
not have a Y
chromosome; or a mixture of subjects that do have a Y chromosome and subjects
that do not
have a Y chromosome.
26. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-25, wherein
the cell is genetically engineered to have reduced expression of Protocadherin-
11 Y-linked
and/or Neuroligin-4 Y-linked using CRISPR/Cas gene editing.
27. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
26, wherein the
CRISPR/Cas gene editing is carried out using one or more guide RNAs comprising
any of the
sequences of Tables 2-5.
28. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-27, wherein
the CRISPR/Cas gene editing is carried out using a Cas effector protein
selected from the group
consisting of Cas9, Cas12a, and Cas12b.
29. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
28, wherein the
292

CRISPR/Cas gene editing is carried out using a Cas effector protein selected
from the group
consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, Casl Od, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cast
0,
Csx11, and Csx10,
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
30. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 26-29,
wherein the CRISPR/Cas gene editing is carried out ex vivo from a donor
subject.
31. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
30, wherein the
CRISPR/Cas gene editing is carried out using a lentiviral vector.
32. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-31, wherein
the cell comprises reduced expression of beta-2-microglobulin (B2M) and/or
MFIC class II
transactivator (CIITA) relative to an unaltered or unmodified wild-type or
control cell.
33. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
32, wherein the cell does
not express B2M and/or CIITA.
34. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-33, wherein
the cell comprises reduced expression of RHD.
293

35. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
34, wherein the cell does
not express REID.
36. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-35, wherein
the cell is a differentiated cell derived from an induced pluripotent stem
cell or a progeny
thereof.
37. The engineered cell of claim 36, wherein the differentiated cell is
selected from
the group consisting of a T cell, a NK cell, an endothelial cell, a pancreatic
islet cell, a cardiac
muscle cell, a smooth muscle cell, a skeletal muscle cell, a hepatocyte, a
glial progenitor cell, a
dopaminergic neuron, a retinal pigment epithelial cell, and a thyroid cell.
38. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-37, wherein
the cell is a primary immune cell or a progeny thereof.
39. The engineered cell of claim 38, wherein the primary immune cell or a
progeny
thereof is a T cell or an NK cell.
40. The engineered cell, hypoimmunogenic T cell, or non-activated T cell of
any one
of claims 1-39, wherein the cell comprises reduced expression of TCR-alpha
and/or TCR-beta.
41. The engineered cell, hypoimmunogenic T cell, or non-activated T cell of
claim
40, wherein the cell does not express TCR-alpha and/or TCR-beta.
42. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 1-41, wherein the cell further comprises a second exogenous
polynucleotide
encoding one or more chimeric antigen receptors (CARs), wherein the one or
more CARs
comprise an extracellular ligand-binding domain having specificity for CD19,
CD20, CD22, or
BCMA, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an
intracellular
signaling domain.
294

43. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 42, wherein the one or more CARs comprise a CD8a hinge domain, a CD28
hinge domain,
or an IgG4 hinge domain.
44. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 43, wherein the one or more CARs comprise a CD8a hinge domain having the
amino acid
sequence of SEQ ID NO: 9.
45. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 43, wherein the one or more CARs comprise a CD28 hinge domain having the
amino acid
sequence of SEQ ID NO: 10 or 113.
46. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 43, wherein the one or more CARs comprise a IgG4 hinge domain having the
amino acid
sequence of SEQ ID NO: 11 or 12.
47. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-46, wherein the one or more CARs comprise a CD8a
transmembrane
domain or a CD28 transmembrane domain.
48. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 47, wherein the one or more CARs comprise a CD8a transmembrane domain
having the
amino acid sequence of SEQ ID NO: 14.
49. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 47, wherein the one or more CARs comprise a CD28 transmembrane domain
having the
amino acid sequence of SEQ ID NO: 15 or 114.
50. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-49, wherein the one or more CARs comprise a 4-1BB
costimulatory
domain, a CD28 costimulatory domain, or a CD3C signaling domain.
295

51. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 50, wherein the one or more CARs comprise a 4-1BB costimulatory domain
having the
amino acid sequence of SEQ ID NO: 16.
52. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 50, wherein the one or more CARs comprise a CD28 costimulatory domain
having the
amino acid sequence of SEQ ID NO: 17.
53. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
claim 50, wherein the one or more CARs comprise a CD3C signaling domain having
the amino
acid sequence of SEQ ID NO: 18 or 115.
54. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-53, wherein the one or more CARs comprise an
extracellular ligand-
binding domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37,
45, 54, 63, 72,
81, or 118, or wherein the CARs have an scFv sequence comprising the heavy and
light chain
sequences of any one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77,
119, or 123.
55. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-54, wherein the one or more CARs have a sequence of any
one of SEQ ID
NOs: 32, 34, 36, 117, or 128.
56. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-55, wherein the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:117 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:117, with
the following
components: CD8a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8a hinge
domain,
CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3C signaling
domain.
57. The engineered cell, hypoimmunogenic T cell, non-activated T cell, or
NK cell of
any one of claims 42-55, wherein the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:45 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
296

least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:45.
58. The engineered cell, hypoimmunogenic T cell, NK cell, non-activated T
cell,
pancreatic islet cell, cardiac muscle cell, glial progenitor cell, or NK cell
of any one of claims 1-
57, wherein one or more of the first and/or second exogenous polynucleotides
is inserted into a
first and/or second specific locus of at least one allele of the cell.
59. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
58, wherein the first
and/or second specific loci are selected from the group consisting of a safe
harbor or target locus,
an RHD locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.
60. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
59, wherein the safe
harbor or target locus is selected from the group consisting of a CCR5 locus,
a CXCR4 locus, a
PPP IR12C locus, an ALB locus, a SH523 I locus, a CLYBL locus, a Rosa locus,
an F3 (CD 142)
locus, a MICA locus, a MICB locus, a LRP I (CD91) locus, a HMGB I locus, an
ABO locus, a
FUT1 locus, and a KDM5D locus.
61. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-60, wherein
the first and/or second exogenous polynucleotide is introduced into the cell
using a gene therapy
vector or a transposase system selected from the group consisting of
transposases, PiggyBac
transposons, Sleeping Beauty (SB11) transposons, Mosl transposons, and To12
transposons.
62. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
61, wherein the gene
therapy vector is a retrovirus or a fusosome
63. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
62, wherein the retrovirus
is a lentiviral vector.
297
CA 03225283 2024- 1- 8

64. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-63, wherein
the first and/or second exogenous polynucleotide is introduced into the cell
using CRISPR/Cas
gene editing.
65. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-64, wherein
the CRISPR/Cas gene editing is carried out using a Cas effector protein
selected from the group
consisting of Cas9, Cas12a, and Cas12b.
66. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
65, wherein the
CRISPR/Cas gene editing is carried out using a Cas effector protein selected
from the group
consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5,
Cas10,
Csx11, and Csx10;
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
67. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 64-66,
wherein the CRISPR/Cas gene editing is carried out ex vivo from a donor
subject.
68. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of claim
67, wherein the
CRISPR/Cas gene editing is carried out using a lentiviral vector.
298
CA 03225283 2024- 1- 8

69. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-68, wherein
the cell or the progeny thereof evades NK cell mediated cytotoxicity upon
administration to a
pati ent.
70. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-69, wherein
the cell or the progeny thereof is protected from cell lysis by mature NK
cells upon
administration to a patient.
71. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-70, wherein
the cell or the progeny thereof evades macrophage engulfment upon
administration to a patient.
72. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-71, wherein
the cell or the progeny thereof does not induce an immune response to the cell
upon
administration to a patient.
73. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-72, wherein
the cell or the progeny thereof does not induce an antibody-based immune
response to the cell
upon administration to a patient.
74. The engineered cell, hypoimmunogenic T cell, non-activated T cell,
pancreatic
islet cell, cardiac muscle cell, glial progenitor cell, or NK cell of any one
of claims 1-73, wherein
the wild-type cell or the control cell is a starting material.
75. A pharmaceutical composition comprising a population of the engineered
cells,
hypoimmunogenic T cells, non-activated T cells, pancreatic islet cells,
cardiac muscle cells, glial
progenitor cell, or NK cells of any one of claims 1-74, and a pharmaceutically
acceptable
additive, carrier, diluent, or excipient.
76. The pharmaceutical composition of claim 75, wherein the composition
comprises
one or more populations of cells selected from the group consisting of a
population of
hypoimmunogenic T cells, a population of non-activated T cells, a population
hypoimmunogenic
299
CA 03225283 2024- 1- 8

CD19 CAR T cells, and a population of hypoimmunogenic CD22 CAR T cells, and a
pharmaceutically acceptable additive, carrier, diluent or excipient.
77. A method of treating a patient with a disease or condition who would
benefit from
a cell-based therapy, comprising administering a population of the engineered
cells,
hypoimmunogenic T cells, non-activated T cells, pancreatic islet cells,
cardiac muscle cells, glial
progenitor cell, or NK cells of any one of claims 1-76 to the patient.
78. The method of claim 77, wherein the patient does not have a Y
chromosome.
79. The method of claim 77 or 78, wherein the patient is not sensitized to
the Y
chromosome gene.
80. The method of claim 77 or 78, wherein the patient is sensitized to the
Y
chromosome gene.
81. The method of claim 80, wherein the patient previously received cell
therapy
derived from a donor subject having a Y chromosome or a cell therapy that
otherwise expressed
one or more of the Y chromosome genes.
82. The method of claim 80 or 81, wherein the patient is a female patient
who was
previously pregnant with a male child.
83. A method of treating cancer in a patient in need thereof comprising
administering
a population of the primary immune cells of any one of claims 1-3 or 7-74 to
the patient.
84. The method of claim 83, wherein the primary immune cells are selected
from the
group consisting of T cells and NK cells.
85. The method of claim 83 or 84, wherein the patient does not have a Y
chromosome.
86. The method of any one of claims 83-85, wherein the patient is not
sensitized to
the Y chromosome gene.
87. The method of any one of claims 83-85, wherein the patient is
sensitized to the Y
chromosome gene.
300
CA 03225283 2024- 1- 8

88. The method of claim 87, wherein the patient previously received cell
therapy
derived from a donor subject having a Y chromosome or a cell therapy that
otherwise expressed
one or more of the Y chromosome genes.
89. The method of claim 87 or 88, wherein the patient is a female patient
who was
previously pregnant with a male child.
90. A method of determining the appropriate cell-based therapy to
administer to a
patient with a disease or condition who would benefit from a cell-based
therapy, comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against one or more Y chromosome genes by:
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NIC
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
301
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligin-4 Y-linked.
91.
A method of identifying a patient with a disease or condition who would
benefit
from a cell-based therapy comprising reduced expression of one or more Y
chromosome genes,
the method comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against one or more Y chromosome genes by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
302
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligi n-4 Y-linked.
92.
A method for identifying a patient with a disease or condition who would
benefit
from a cell-based therapy comprising reduced expression of Protocadherin-11 Y-
linked and/or of
Neuroligin-4 Y-linked, the method comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
303
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligin-4 Y-linked.
93. A method of determining whether a cell-based therapy that
does not comprise
reduced expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-
linked is susceptible
to NK mediated cytotoxicity upon administration to a patient, the method
comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
304
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligi n-4 Y-linked.
94. A method of determining whether a cell-based therapy that
does not comprise
reduced expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-
linked is susceptible
to lysis by mature NK cells upon administration to a patient, the method
comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
305
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligi n-4 Y-linked.
95. A method of determining whether a cell-based therapy that
does not comprise
reduced expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-
linked is susceptible
to macrophage engulfment upon administration to a patient, the method
comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
306
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligi n-4 Y-linked.
96. A method of determining whether a cell-based therapy that
does not comprise
reduced expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-
linked is susceptible
to an induced immune response upon administration to a patient, the method
comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by.
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
307
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligin-4 Y-linked.
97. A method of determining whether a cell-based therapy that
does not comprise
reduced expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-
linked is susceptible
to an induced antibody-based immune response upon administration to a patient,
the method
comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against Protocadherin-11 Y-linked and/or antibodies against Neuroligin-4 Y-
linked by:
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
308
CA 03225283 2024- 1- 8

(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligi n-4 Y-linked.
98.
A method of treating a patient with a disease or condition who would
benefit from
a cell-based therapy, comprising:
(a) determining whether a biological sample from the patient comprises
antibodies
against one or more Y chromosome genes by:
(i) obtaining or having obtained a biological sample from the patient;
(ii) performing or having performed an assay to determine whether antibodies
against Protocadherin-11 Y-linked are present in the biological sample; and
(iii) performing or having performed an assay to determine whether antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and
(b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells of any one of claims 1-74 to the patient, wherein:
(i) if antibodies against Protocadherin-11 Y-linked are present in the
biological
sample, the population of cells comprises reduced expression of Protocadherin-
11 Y-linked;
(ii) if antibodies against Neuroligin-4 Y-linked are present in the biological

sample, the population of cells comprises reduced expression of Neuroligin-4 Y-

linked;
(iii) if antibodies against Protocadherin-11 Y-linked and antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked;
(iv) if neither antibodies against Protocadherin-11 Y-linked nor antibodies
against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells
309
CA 03225283 2024- 1- 8

does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligin-4 Y-linked.
99.
The method of any one of claims 77-98, wherein the Y chromosome gene is a
Y
chromosome linked antigen or a minor histocompatibility antigen associated
with the Y
chromosome.
100. The method of claim 99, wherein the one or more Y chromosome linked
antigens
are Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
101. The method of any one of claims 77-100, wherein the cell has reduced
expression
of Protocadherin-11 Y-linked.
102. The method of any one of claims 77-101, wherein the cell has reduced
expression
of Neuroligin-4 Y-linked.
103. The method of any one of claims 77-102, wherein the cell has reduced
expression
of Protocadherin-11 Y-linked and reduced expression of Neuroligin-4 Y-linked.
104. The method of any one of claims 77-103, wherein the cell is genetically
engineered to have reduced expression of Protocadherin-11 Y-linked and/or
Neuroligin-4 Y-
linked.
105. The method of any one of claims 77-104, wherein the cell does not express

Protocadherin-11 Y-linked.
106. The method of any one of claims 77-105, wherein the cell does not express

Neuroligin-4 Y-linked.
107. The method of any one of claims 77-106, wherein the cell does not express

Protocadherin-11 Y-linked and does not express Neuroligin-4 Y-linked.
108. The method of any one of claims 77-107, wherein the cell is genetically
engineered to not express Protocadherin-11 Y-linked and/or Neuroligin-4 Y-
linked.
109. The method of any one of claims 77-108, wherein reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked is caused by a knock
out of the
PCDH11Y and/or NLGN4Y gene, respectively.
310
CA 03225283 2024- 1- 8

110. The method of any one of claims 77-109, wherein the cell is derived from
a
human cell or an animal cell.
111. The method of claim 110, wherein the human cell or animal cell is from a
donor
subject that does not have a Y chromosome.
112. The method of claim 110, wherein the human cell or animal cell is from a
donor
subject that has a Y chromosome, and wherein the cell is genetically
engineered to have reduced
expression of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
113. The method of claim 112, wherein the cell is genetically engineered to
not express
Protocadherin-11 Y-linked.
114. The method of claim 112, wherein the cell is genetically engineered to
not express
Neuroligin-4 Y-linked.
115. The method of claim 112, wherein the cell is genetically engineered to
not express
Protocadherin-11 Y-linked and to not express Neuroligin-4 Y-linked.
116. The method of any one of claims 77-115, wherein the cell is propagated or

derived from a pool of cells that are isolated from one or more donor subjects
different from the
patient, wherein the one or more donor subjects optionally comprise one or
more subjects that
have a Y chromosome; one or more subjects that do not have a Y chromosome; or
a mixture of
subjects that do have a Y chromosome and subjects that do not have a Y
chromosome.
117. The method of any one of claims 77-116, wherein the cell is genetically
engineered to have reduced expression of Protocadherin-11 Y-linked and/or
Neuroligin-4 Y-
linked using CRISPR/Cas gene editing.
118. The method of claim 117, wherein the CRISPR/Cas gene editing is carried
out
using one or more guide RNAs comprising any of the sequences of Tables 2-5.
119. The method of any one of claims 117-118, wherein the CRISPR/Cas gene
editing
is carried out using a Cas effector protein selected from the group consisting
of Cas9, Cas12a,
and Cas12b.
311
CA 03225283 2024- 1- 8

120. The method of claim 119, wherein the CRISPR/Cas gene editing is carried
out
using a Cas effector protein selected from the group consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5,
Cas10,
Csx11, and Csx10,
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
121. The method of any one of claims 117-120, wherein the CRISPR/Cas gene
editing
is carried out ex vivo from a donor subject.
122. The method of claim 121, wherein the CRISPRICas gene editing is carried
out
using a lentiviral vector.
123. The method of any one of claims 77-122, wherein the cell comprises
reduced
expression of B2M and/or CIITA relative to an unaltered or unmodified wild-
type or control cell.
124. The method of claim 123, wherein the cell does not express B2M and/or
CIITA.
125. The method of any one of claims 77-124, wherein the cell comprises
reduced
expression of RHD.
126. The method of claim 125, wherein the cell does not express RHD.
127. The method of any one of claims 77-126, wherein the cell is a
differentiated cell
derived from an induced pluripotent stem cell or a progeny thereof.
128. The method of claim 127, wherein the differentiated cell is selected from
the
group consisting of a T cell, a NK cell, an endothelial cell, a pancreatic
islet cell, a cardiac
muscle cell, a smooth muscle cell, a skeletal muscle cell, a hepatocyte, a
glial progenitor cell, a
dopaminergic neuron, a retinal pigment epithelial cell, and a thyroid cell.
312
CA 03225283 2024- 1- 8

129. The method of any one of claims 77-126, wherein the cell is a primary
immune
cell or a progeny thereof.
130. The engineered cell of claim 129, wherein the primary immune cell or a
progeny
thereof is a T cell or an NK cell.
131. The method of any one of claims 77-130, wherein the cell comprises
reduced
expression of TCR-alpha and/or TCR-beta.
132. The method of claim 131, wherein the cell does not express TCR-alpha
and/or
TCR-beta.
133. The method of any one of claims 77-132, wherein the cell further
comprises a
second exogenous polynucleotide encoding one or more CARs, wherein the one or
more CARs
comprise an extracellular ligand-binding domain having specificity for CD19,
CD20, CD22, or
BCMA, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an
intracellular
signaling domain.
134. The method of claim 133, wherein the one or more CARs comprise a CD8a
hinge
domain, a CD28 hinge domain, or an IgG4 hinge domain.
135. The method of claim 134, wherein the one or more CARs comprise a CD8a
hinge
domain having the amino acid sequence of SEQ ID NO: 9.
136. The method of claim 134, wherein the one or more CARs comprise a CD28
hinge
domain having the amino acid sequence of SEQ ID NO: 10 or 113.
137. The method of claim 134, wherein the one or more CARs comprise a IgG4
hinge
domain having the amino acid sequence of SEQ ID NO: 11 or 12.
138. The method of any one of claims 133-137, wherein the one or more CARs
comprise a CD8a transmembrane domain or a CD28 transmembrane domain.
139. The method of claim 138, wherein the one or more CARs comprise a CD8a
transmembrane domain having the amino acid sequence of SEQ ID NO: 14.
313
CA 03225283 2024- 1- 8

140. The method of claim 138, wherein the one or more CARs comprise a CD28
transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.
141. The method of any one of claims 133-140, wherein the one or more CARs
comprise a 4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3C
signaling
domain.
142. The method of claim 141, wherein the one or more CARs comprise a 4-1BB
costimulatory domain having the amino acid sequence of SEQ ID NO: 16.
143. The method of claim 141, wherein the one or more CARs comprise a CD28
costimulatory domain having the amino acid sequence of SEQ ID NO: 17.
144. The method of claim 141, wherein the one or more CARs comprise a CD3t.
signaling domain having the amino acid sequence of SEQ ID NO: 18 or 115.
145. The method of any one of claims 133-144, wherein the one or more CARs
comprise an extracellular ligand-binding domain comprising an scFv sequence of
any one of
SEQ ID NOs: 19, 37, 45, 54, 63, 72, 81, or 118, or wherein the CARs have an
scFv sequence
comprising the heavy and light chain sequences of any one of SEQ ID NOs: 20,
25, 38, 42, 46,
50, 64, 68, 73, 77, 119, or 123.
146. The method of any one of claims 133-145, wherein the one or more CARs
have a
sequence of any one of SEQ NOs: 32, 34, 36, 117, or 128.
147. The method of any one of claims 133-146, wherein the one or more CARs
comprise an amino acid sequence set forth in SEQ ID NO:117 or an amino acid
sequence at least
80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least
97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence
set forth in of
SEQ ID NO:117, with the following components: CD8a signal peptide, FMC63 scFv
(VL-
Whitlow linker-VH), CD8a hinge domain, CD8a transmembrane domain, 4-1BB
costimulatory
domain, and CD3C signaling domain.
314
CA 03225283 2024- 1- 8

148. The method of any one of claims 133-146, wherein the one or more CARs
comprise an amino acid sequence set forth in SEQ ID NO:45 or an amino acid
sequence at least
80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least
97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence
set forth in of
SEQ ID NO:45.
149. The method of any one of claims 77-148, wherein one or more of the first
and/or
second exogenous polynucleotides is inserted into a first and/or second
specific locus of at least
one allele of the cell.
150. The method of claim 149, wherein the first and/or second specific loci
are
selected from the group consisting of a safe harbor or target locus, an RHD
locus, a B2114- locus, a
CHTA locus, a TRAC locus, and a TRB locus.
151. The method of claim 150, wherein the safe harbor or target locus is
selected from
the group consisting of a CCR5 locus, a CXCR4 locus, a PPP I R12C locus, an
ALB locus, a
SHS231 locus, a CLYBL locus, a Rosa locus, an F3 (CD142) locus, a MICA locus,
a MICB locus,
a LRP 1 (CD91) locus, a HMGB 1 locus, an ABO locus, a FUT] locus, and a KDM5D
locus.
152. The method of any one of claims 77-151, wherein the first and/or second
exogenous polynucleotide is introduced into the cells using a gene therapy
vector or a
transposase system selected from the group consisting of transposases,
PiggyBac transposons,
Sleeping Beauty (SB11) transposons, Mosl transposons, and To12 transposons.
153. The method of claims 152, wherein the gene therapy vector is a retrovirus
or a
fusosome.
154. The method of claim 153, wherein the retrovirus is a lentiviral vector.
155. The method of any one of claims 77-154, wherein the first and/or second
exogenous polynucleotide is introduced into the cell using CRISPR/Cas gene
editing.
156. The method of any one of claims 77-155, wherein the CRISPR/Cas gene
editing
is carried out using a Cas effector protein selected from the group consisting
of Cas9, Cas12a,
and Cas12b.
315
CA 03225283 2024- 1- 8

157. The method of claim 156, wherein the CRISPR/Cas gene editing is carried
out
using a Cas effector protein selected from the group consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cast
0,
Csx11, and Csx10,
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
158. The method of any one of claims 155-157, wherein the CRISPR/Cas gene
editing
is carried out ex vivo from a donor subject.
159. The method of claim 158, wherein the CRISPRICas gene editing is carried
out
using a lentiviral vector.
160. The method of any one of claims 77-159, wherein the cell or the progeny
thereof
evades NK cell mediated cytotoxicity upon administration to a patient.
161. The method of any one of claims 77-160, wherein the cell or the progeny
thereof
is protected from cell lysis by mature NK cells upon administration to a
patient.
162. The method of any one of claims 77-161, wherein the cell or the progeny
thereof
evades macrophage engulfment upon administration to a patient.
163. The method of any one of claims 77-162, wherein the cell or the progeny
thereof
does not induce an immune response to the cell upon administration to a
patient.
164. The method of any one of claims 77-163, wherein the cell or the progeny
thereof
does not induce an antibody-based immune response to the cell upon
administration to a patient.
165. The method of any one of claims 77-164, wherein the wild-type cell or the
control
cell is a starting material.
316
CA 03225283 2024- 1- 8

166. Use of a population of engineered T cells for treating a disorder or
condition in a
patient, wherein the engineered T cells comprise reduced expression of one or
more Y
chromosome genes and MEC class I and/or class II human leukocyte antigen
molecules relative
to an unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide
encoding CD47, wherein the engineered T cells are propagated from a primary T
cell or a
progeny thereof, or are derived from an iPSC or a progeny thereof.
167. Use of a population of engineered differentiated cells for treating a
disorder or
condition in a patient, wherein the engineered differentiated cells comprise
reduced expression of
one or more Y chromosome genes and MI-IC class I and/or class II human
leukocyte antigen
molecules relative to an unaltered or unmodified wild-type or control cell,
and a first exogenous
polynucleotide encoding CD47, wherein the engineered differentiated cells are
derived an iPSC
or a progeny thereof.
168. The use of claim 166 or 167, wherein the Y chromosome gene is a Y
chromosome
linked antigen or a minor histocompatibility antigen associated with the Y
chromosome.
169. The use of claim 168, wherein the one or more Y chromosome linked
antigens are
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
170. The use of any one of claims 166-169, wherein the cell has reduced
expression of
Protocadherin-11 Y-linked.
171. The use of any one of claims 166-170, wherein the cell has reduced
expression of
Neuroligin-4 Y-linked.
172. The use of any one of claims 166-171, wherein the cell has reduced
expression of
Protocadherin-11 Y-linked and reduced expression of Neuroligin-4 Y-linked.
173. The use of any one of claims 166-172, wherein the cell is genetically
engineered to
have reduced expression of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-
linked.
174. The use of any one of claims 166-173, wherein the cell does not express
Protocadherin-11 Y-linked.
175. The use of any one of claims 166-174, wherein the cell does not express
Neuroligin-4 Y-linked.
317
CA 03225283 2024- 1- 8

176. The use of any one of claims 166-175, wherein the cell does not express
Protocadherin-11 Y-linked and does not express Neuroligin-4 Y-linked.
177. The use of any one of claims 166-176, wherein the cell is genetically
engineered to
not express Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
178. The use of any one of claims 166-177, wherein reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked is caused by a knock
out of the
PCDH11Y and/or NLGN4Y gene, respectively.
179. The use of any one of claims 166-178, wherein the cell is derived from a
human cell
or an animal cell.
180. The use of claim 179, wherein the human cell or animal cell is from a
donor subject
that does not have a Y chromosome.
181. The use of claim 179, wherein the human cell or animal cell is from a
donor subject
that has a Y chromosome, and wherein the cell is genetically engineered to
have reduced
expression of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
182. The use of claim 181, wherein the cell is genetically engineered to not
express
Protocadherin-11 Y-linked.
183. The use of claim 181, wherein the cell is genetically engineered to not
express
Neuroligin-4 Y-linked.
184. The use of claim 181, wherein the cell is genetically engineered to not
express
Protocadherin-11 Y-linked and to not express Neuroligin-4 Y-linked.
185. The use of any one of claims 166-184, wherein the cell is propagated or
derived
from a pool of cells that are isolated from one or more donor subjects
different from the patient,
wherein the one or more donor subjects optionally comprise one or more
subjects that have a Y
chromosome; one or more subjects that do not have a Y chromosome; or a mixture
of subjects
that do have a Y chromosome and subjects that do not have a Y chromosome.
186. The use of any one of claims 166-185, wherein the cell is genetically
engineered to
have reduced expression of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-
linked using
CRISPR/Cas gene editing.
318
CA 03225283 2024- 1- 8

187. The use of claim 186, wherein the CRISPR/Cas gene editing is carried out
using
one or more guide RNAs comprising any of the sequences of Tables 2-5.
188. The use of any one of claims 186-187, wherein the CRISPR/Cas gene editing
is
carried out using a Cas effector protein selected from the group consisting of
Cas9, Cas12a, and
Cas12b.
189. The use of claim 188, wherein the CRISPR/Cas gene editing is carried out
using a
Cas effector protein selected from the group consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5,
Cas10,
Csx11, and Csx10;
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
190. The use of any one of claims 186-189, wherein the CRISPR/Cas gene editing
is
carried out ex vivo from a donor subject.
191. The use of claim 144, wherein the CRISPR/Cas gene editing is carried out
using a
lentiviral vector.
192. The use of any one of claims 166-191, wherein the cell comprises reduced
expression of B2M and/or CIITA relative to an unaltered or unmodified wild-
type or control cell.
193. The use of claim 192, wherein the cell does not express B2M and/or CIITA.
194. The use of any one of claims 166-193, wherein the cell comprises reduced
expression of RHD.
195. The use of claim 194, wherein the cell does not express RHD.
319
CA 03225283 2024- 1- 8

196. The use of any one of claims 166-195, wherein the cell is a
differentiated cell
derived from an induced pluripotent stem cell or a progeny thereof.
197. The use of claim 196, wherein the differentiated cell is selected from
the group
consisting of a T cell, a NK cell, an endothelial cell, a pancreatic islet
cell, a cardiac muscle cell,
a smooth muscle cell, a skeletal muscle cell, a hepatocyte, a glial progenitor
cell, a dopaminergic
neuron, a retinal pigment epithelial cell, and a thyroid cell.
198. The use of any one of claims 166-195, wherein the cell is a primary
immune cell or
a progeny thereof.
199. The use of claim 198, wherein the primary immune cell or a progeny
thereof is a T
cell or an NK cell.
200. The use of any one of claims 166-199, wherein the cell comprises reduced
expression of TCR-alpha and/or TCR-beta.
201. The use of claim 200, wherein the cell does not express TCR-alpha and/or
TCR-
beta.
202. The use of any one of claims 166-201, wherein the cell further comprises
a
second exogenous polynucleotide encoding one or more CARs, wherein the one or
more CARs
comprise an extracellular ligand-binding domain having specificity for CD19,
CD20, CD22, or
BCMA, a hinge domain, a transmembrane domain, a co-stimulatory domain, and an
intracellular
signaling domain.
203. The use of claim 202, wherein the one or more CARs comprise a CD8a hinge
domain, a CD28 hinge domain, or an IgG4 hinge domain.
204. The use of claim 203, wherein the one or more CARs comprise a CD8a hinge
domain having the amino acid sequence of SEQ ID NO: 9.
205. The use of claim 203, wherein the one or more CARs comprise a CD28 hinge
domain having the amino acid sequence of SEQ ID NO: 10 or 113.
206. The use of claim 203, wherein the one or more CARs comprise a IgG4 hinge
domain having the amino acid sequence of SEQ ID NO: 11 or 12.
320
CA 03225283 2024- 1- 8

207. The use of any one of claims 202-206, wherein the one or more CARs
comprise a
CD8a transmembrane domain or a CD28 transmembrane domain.
208. The use of claim 207, wherein the one or more CARs comprise a CD8a
transmembrane domain having the amino acid sequence of SEQ ID NO: 14.
209. The use of claim 207, wherein the one or more CARs comprise a CD28
transmembrane domain having the amino acid sequence of SEQ ID NO: 15 or 114.
210. The use of any one of claims 202-209, wherein the one or more CARs
comprise a
4-1BB costimulatory domain, a CD28 costimulatory domain, or a CD3C signaling
domain.
211. The use of claim 210, wherein the one or more CARs comprise a 4-1BB
costimulatory domain having the amino acid sequence of SEQ ID NO: 16.
212. The use of claim 210, wherein the one or more CARs comprise a CD28
costimulatory domain having the amino acid sequence of SEQ ID NO: 17.
213. The use of claim 210, wherein the one or more CARs comprise a CD3
signaling
domain having the amino acid sequence of SEQ ID NO: 18 or 115.
214. The use of any one of claims 202-213, wherein the one or more CARs
comprise
an extracellular ligand-binding domain comprising an scFv sequence of any one
of SEQ ID NOs:
19, 37, 45, 54, 63, 72, 81, or 118, or wherein the CARs have an scFv sequence
comprising the
heavy and light chain sequences of any one of SEQ ID NOs: 20, 25, 38, 42, 46,
50, 64, 68, 73,
77, 119, or 123.
215. The use of any one of claims 202-214, wherein the one or more CARs have a
sequence of any one of SEQ NOs: 32, 34, 36, 117, or 128.
216. The use of any one of claims 202-215, wherein the one or more CARs
comprise
an amino acid sequence set forth in SEQ ID NO:117 or an amino acid sequence at
least 80%
identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%,
at least 98%, at least 99%, or 100% identical) to the amino acid sequence set
forth in of SEQ ID
NO:117, with the following components: CD8a signal peptide, FMC63 scFv (VL-
Whitlow
321
CA 03225283 2024- 1- 8

linker-VH), CD8a hinge domain, CD8a transmembrane domain, 4- 1BB costimulatory
domain,
and CD3C signaling domain.
217. The use of any one of claims 202-216, wherein the one or more CARs
comprise an
amino acid sequence set forth in SEQ ID NO:45 or an amino acid sequence at
least 80%
identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%,
at least 98%, at least 99%, or 100% identical) to the amino acid sequence set
forth in of SEQ ID
NO:45.
218. The use of any one of claims 166-217, wherein one or more of the first
and/or
second exogenous polynucleotides is inserted into a first and/or second
specific locus of at least
one allele of the cell.
219. The use of claim 218, wherein the first and/or second specific loci are
selected from
the group consisting of a safe harbor or target locus, an RHD locus, a B2M
locus, a CHTA locus,
a TRAC locus, and a TRB locus.
220. The use of claim 219, wherein the safe harbor or target locus is selected
from the
group consisting of a CCR5 locus, a CXCR4 locus, a PPP 1R12C locus, an ALB
locus, a SHS231
locus, a CLYBL locus, a Rosa locus, an F3 (CD 142) locus, a MICA locus, a MICB
locus, a LRP 1
(CD91) locus, a II1V1GB1 locus, an ABO locus, a FUT1 locus, and a KD1V15D
locus.
221. The use of any one of claims 166-220, wherein the first and/or second
exogenous
polynucleotide is introduced into the engineered T cells using a gene therapy
vector or a
transposase system selected from the group consisting of transposases,
PiggyBac transposons,
Sleeping Beauty (SB11) transposons, Mosl transposons, and To12 transposons.
222. The use of claim 221, wherein the gene therapy vector is a retrovirus or
a
fusosome.
223. The use of claim 222, wherein the retrovirus is a lentiviral vector.
224. The use of any one of claims 166-223, wherein the first and/or second
exogenous
polynucleotide is introduced into the cell using CRISPR/Cas gene editing.
322
CA 03225283 2024- 1- 8

225. The use of any one of claims 177-224, wherein the CRISPR/Cas gene editing
is
carried out using a Cas effector protein selected from the group consisting of
Cas9, Cas12a, and
Cas12b.
226. The use of claim 225, wherein the CRISPR/Cas gene editing is carried out
using a
Cas effector protein selected from the group consisting of:
a. optionally selected from the group consisting of Cas3, Cas8a, Cas5,
Cas8b,
Cas8c, CaslOd, Csel, Cse2, Csy 1, Csy2, Csy3, and GSU00.54;
b. optionally selected from the group consisting of Cas9, Csn2, and Cas4;
c. optionally selected from the group consisting of Cas10, Csm2, Cmr5, Cast
0,
Csx11, and Csx10;
d. optionally Csfl;
e. optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4,
C2c8, C2c5, C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and
f. optionally selected from the group consisting of Cas13, Cas13a, C2c2,
Cas13b,
Cas13c, and Cas13d.
227. The use of any one of claims 224-226, wherein the CRISPR/Cas gene editing
is
carried out ex vivo from a donor subject.
228. The use of claim 227, wherein the CRISPR/Cas gene editing is carried out
using a
lentiviral vector.
229. The use of any one of claims 166-228, wherein the cell or the progeny
thereof
evades NK cell mediated cytotoxicity upon administration to a patient.
230. The use of any one of claims 166-229, wherein the cell or the progeny
thereof is
protected from cell lysis by mature NK cells upon administration to a patient.
231. The use of any one of claims 166-230, wherein the cell or the progeny
thereof
evades macrophage engulfment upon administration to a patient.
232. The use of any one of claims 166-231, wherein the cell or the progeny
thereof does
not induce an immune response to the cell upon administration to a patient.
323
CA 03225283 2024- 1- 8

233. The use of any one of claims 166-232, wherein the cell or the progeny
thereof does
not induce an antibody-based immune response to the cell upon administration
to a patient.
234. The method of any one of claims 177-233, wherein the wild-type cell or
the control
cell is a starting material.
235. A method for producing an engineered cell comprising reduced expression
of one
or more Y chromosome genes and MI-IC class I and/or class II human leukocyte
antigen
molecules relative to an unaltered or unmodified wild-type or control cell,
and a first exogenous
polynucleotide encoding CD47, the method comprising:
(a) obtaining an isolated cell;
(b) genetically modifying the cell to reduce expression of the one or more Y
chromosome
genes in the cell;
(c) genetically modifying the cell to reduce expression of MI-IC class I human
leukocyte
antigen molecules and/or MHC class II human leukocyte antigen molecules in the
cell;
and
(d) introducing into the isolated cell a polynucleotide encoding CD47, to
thereby produce
the engineered cell.
236. A method for producing an engineered cell comprising reduced expression
of one
or more Y chromosome genes and IVIRC class I and/or class II human leukocyte
antigen
molecules relative to an unaltered or unmodified wild-type or control cell,
and a first exogenous
polynucleotide encoding CD47, the method comprising:
(a) obtaining an isolated cell;
(b) contacting the cell with a composition comprising lentiviral vectors
comprising
(i) a CD4 binding agent or a CD8 binding agent,
(ii) polynucleotides encoding CRISPR/Cas gene editing components targeting the

one or more Y chromosome gene loci,
(iii) polynucleotides encoding CRISPR/Cas gene editing components targeting
the
MFIC class I and/or class II human leukocyte antigen gene loci, and
324
CA 03225283 2024- 1- 8

(iv) a first exogenous polynucleotide encoding CD47, to thereby produce the
engineered cell.
325
CA 03225283 2024- 1- 8

Description

WO 2023/287827
PCT/US2022/036874
ALTERED EXPRESSION OF Y CHROMOSOME-LINKED ANTIGENS IN
HYPOIMMUNOGENIC CELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application claims priority to U.S. Provisional Application No.
63/221,887, filed
July 14, 2021, and U.S. Provisional Application No. 63/255,914, filed October
14, 2021, the
disclosure of each of which is herein incorporated in its entirety.
BACKGROUND
100021 Off-the-shelf CAR-T cells and other therapeutic cells can offer
advantages over
autologous cell-based strategies, including ease of manufacturing, quality
control and avoidance
of malignant contamination and T cell dysfunction. However, the vigorous host-
versus-graft
immune response against histoincompatible T cells prevents expansion and
persistence of
allogeneic CAR-T cells and mitigates the efficacy of this approach.
100031 There is substantial evidence in both animal models and human patients
that
hypoimmunogenic cell transplantation is a scientifically feasible and
clinically promising
approach to the treatment of numerous disorders, conditions, and diseases.
100041 There remains a need for novel approaches, compositions and methods for
producing
cell-based therapies that avoid detection by the recipient's immune system.
SUMMARY
100051 In some embodiments, provided herein is an engineered cell comprising
reduced
expression of one or more Y chromosome genes and major histocompatibility
complex (MHC)
class I and/or class II human leukocyte antigen molecules relative to an
unaltered or unmodified
wild-type or control cell, and a first exogenous polynucleotide encoding CD47,
wherein the
engineered cell is propagated from a primary T cell or a progeny thereof, or
is derived from an
induced pluripotent stem cell (iPSC) or a progeny thereof.
100061 In some embodiments, provided herein is a hypoimmunogenic T cell
comprising
reduced expression of one or more Y chromosome genes and MHC class I and/or
class II human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the hypoimmunogenic T
cell is
1
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
propagated from a primary T cell or a progeny thereof, or is derived from an
iPSC or a progeny
thereof.
100071 In some embodiments, provided herein is a non-activated T cell
comprising reduced
expression of one or more Y chromosome genes and MHC class I and/or class II
human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the non-activated T
cell is propagated
from a primary T cell or a progeny thereof, or is derived from an iPSC or a
progeny thereof.
100081 In some embodiments, provided herein is a pancreatic islet cell
comprising reduced
expression of one or more Y chromosome genes and MHC class I and/or class II
human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the pancreatic islet
cell is derived
from an iPSC or a progeny thereof.
100091 In some embodiments, provided herein is a cardiac muscle cell
comprising reduced
expression of one or more Y chromosome genes and MHC class I and/or class II
human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the cardiac muscle
cell is derived
from an iPSC or a progeny thereof.
100101 In some embodiments, provided herein is a glial progenitor cell
comprising reduced
expression of one or more Y chromosome genes and MHC class I and/or class II
human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the cardiac muscle
cell is derived
from an iPSC or a progeny thereof.
10011] In some embodiments, provided herein is a NK cell comprising reduced
expression of
one or more Y chromosome genes and MEW class I and/or class II human leukocyte
antigen
molecules relative to an unaltered or unmodified wild-type or control cell,
and a first exogenous
polynucleotide encoding CD47, wherein the cardiac muscle cell is derived from
an iPSC or a
progeny thereof.
100121 In some embodiments, the Y chromosome gene is a Y chromosome linked
antigen or a
minor histocompatibility antigen associated with the Y chromosome.
2
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[0013] In some embodiments, the one or more Y chromosome linked antigens are
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
100141 In some embodiments, the cell has reduced expression of Protocadherin-
11 Y-linked.
[0015] In some embodiments, the cell has reduced expression of Neuroligin-4 Y-
linked.
100161 In some embodiments, the cell has reduced expression of Protocadherin-
11 Y-linked
and reduced expression of Neuroligin-4 Y-linked.
[0017] In some embodiments, the cell is genetically engineered to have reduced
expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
[0018] In some embodiments, the cell does not express Protocadherin-11 Y-
linked.
[0019] In some embodiments, the cell does not express Neuroligin-4 Y-linked.
[0020] In some embodiments, the cell does not express Protocadherin-11 Y-
linked and does
not express Neuroligin-4 Y-linked.
[0021] In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked and/or Neuroligin-4 Y-linked.
[0022] In some embodiments, reduced expression of Protoeadherin-11 Y-linked
and/or
Neuroligin-4 Y-linked is caused by a knock out of the PCDH11Y and/or NI_GN4Y
gene,
respectively.
[0023] In some embodiments, the cell is derived from a human cell or an animal
cell.
[0024] In some embodiments, the human cell or animal cell is from a donor
subject that does
not have a Y chromosome.
[0025] In some embodiments, the human cell or animal cell is from a donor
subject that has a
Y chromosome, and the cell is genetically engineered to have reduced
expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
100261 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked.
[0027] In some embodiments, the cell is genetically engineered to not express
Neuroligin-4 Y-
linked.
3
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[0028] In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked and to not express Neuroligin-4 Y-linked.
100291 In some embodiments, the cell is propagated or derived from a pool of
cells that are
isolated from one or more donor subjects different from the patient, and the
one or more donor
subjects optionally comprise one or more subjects that have a Y chromosome;
one or more
subjects that do not have a Y chromosome; or a mixture of subjects that do
have a Y
chromosome and subjects that do not have a Y chromosome.
[0030] In some embodiments, the cell is genetically engineered to have reduced
expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked using CRISPR/Cas gene
editing.
[0031] In some embodiments, the CRISPR/Cas gene editing is carried out using
one or more
guide RNAs comprising any of the sequences of Tables 2-5.
100321 In some embodiments, the CRISPR/Cas gene editing is carried out using a
Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b.
[0033] In some embodiments, the CRISPR/Cas gene editing is carried out using a
Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d)
optionally Csfl,
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected
from the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.
[0034] In some embodiments, the CRISPR/Cas gene editing is carried out ex vivo
from a
donor subject.
[0035] In some embodiments, the CRISPR/Cas gene editing is carried out using a
lentiyiral
vector.
[0036] In some embodiments, the cell comprises reduced expression of beta-2-
microglobulin
(B2M) and/or MEW class II transactivator (CIITA) relative to an unaltered or
unmodified wild-
type or control cell.
4
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[0037] In some embodiments, the cell does not express B2M and/or CIITA.
[0038] In some embodiments, the cell comprises reduced expression of RHD.
[0039] In some embodiments, the cell does not express RHD.
100401 In some embodiments, the cell is a differentiated cell derived from an
induced
pluripotent stem cell or a progeny thereof
[0041] In some embodiments, the differentiated cell is selected from the group
consisting of a
T cell, a NK cell, an endothelial cell, a pancreatic islet cell, a cardiac
muscle cell, a smooth
muscle cell, a skeletal muscle cell, a hepatocyte, a glial progenitor cell, a
dopaminergic neuron, a
retinal pigment epithelial cell, and a thyroid cell.
[0042] In some embodiments, the cell is a primary immune cell or a progeny
thereof.
[0043] In some embodiments, the primary immune cell or a progeny thereof is a
T cell or an
NK cell.
[0044] In some embodiments, the cell comprises reduced expression of TCR-alpha
and/or
TCR-beta.
[0045] In some embodiments, the cell does not express TCR-alpha and/or TCR-
beta.
[0046] In some embodiments, the cell further comprises a second exogenous
polynucleotide
encoding one or more chimeric antigen receptors (CARs), and the one or more
CARs comprise
an extracellular ligand-binding domain having specificity for CD19, CD20,
CD22, or BCMA, a
hinge domain, a transmembrane domain, a co-stimulatory domain, and an
intracellular signaling
domain.
[0047] In some embodiments, the one or more CARs comprise a CD8ct hinge
domain, a CD28
hinge domain, or an IgG4 hinge domain.
100481 In some embodiments, the one or more CARs comprise a CD8ct hinge domain
having
the amino acid sequence of SEQ ID NO: 9.
[0049] In some embodiments, the one or more CARs comprise a CD28 hinge domain
having
the amino acid sequence of SEQ ID NO: 10 or 113.
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[0050] In some embodiments, the one or more CARs comprise a IgG4 hinge domain
having
the amino acid sequence of SEQ ID NO: 11 or 12.
100511 In some embodiments, the one or more CARs comprise a CD8a transmembrane

domain or a CD28 transmembrane domain.
[0052] In some embodiments, the one or more CARs comprise a CD8a transmembrane

domain having the amino acid sequence of SEQ ID NO: 14.
[0053] In some embodiments, the one or more CARs comprise a CD28 transmembrane
domain
having the amino acid sequence of SEQ ID NO: 15 or 114.
[0054] In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain,
a CD28 costimulatory domain, or a CD3t signaling domain.
[0055] In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain
having the amino acid sequence of SEQ ID NO: 16.
100561 In some embodiments, the one or more CARs comprise a CD28 costimulatory
domain
having the amino acid sequence of SEQ ID NO: 17.
[0057] In some embodiments, the one or more CARs comprise a CD3C signaling
domain
having the amino acid sequence of SEQ ID NO: 18 or 115,
[0058] In some embodiments, the one or more CARs comprise an extracellular
ligand-binding
domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54,
63, 72, 81, or
118, or and the CARs have an scFy sequence comprising the heavy and light
chain sequences of
any one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.
[0059] In some embodiments, the one or more CARs have a sequence of any one of
SEQ ID
NOs: 32, 34, 36, 117, or 128.
[0060] In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:117 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:117, with
the following
components: CD8a signal peptide, FMC63 scFy (VL-Whitlow linker-VH), CD8a,
hinge domain,
CD8a transmembrane domain, 4-BB costimulatory domain, and CD3 signaling
domain.
6
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
100611 In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:45 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:45.
100621 In some embodiments, one or more of the first and/or second exogenous
polynucleotides is inserted into a first and/or second specific locus of at
least one allele of the
cell.
100631 In some embodiments, the first and/or second specific loci are selected
from the group
consisting of a safe harbor or target locus, an RHD locus, a B2M locus, a
CIITA locus, a TRAC
locus, and a TRB locus.
100641 In some embodiments, the safe harbor or target locus is selected from
the group
consisting of a CCR5 locus, a CXCR./ locus, a
1 R12C locus, an ALB locus, a SHS'231 locus,
a CIYBI locus, a Rosa locus, an F3 (CD 142) locus, a MICA locus, a MICR locus,
a 1RP 1
(CD91) locus, a HMGB 1 locus, an ABO locus, a FUT 1 locus, and a KDM5D locus.
100651 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the cell using a gene therapy vector or a transposase system selected
from the group
consisting of transposases, PiggyBac transposons, Sleeping Beauty (SB11)
transposons, Mosl
transposons, and To12 transposons.
100661 In some embodiments, the gene therapy vector is a retrovirus or a
fusosome.
100671 In some embodiments, the retrovirus is a lentiviral vector.
100681 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the cell using CRISPR/Cas gene editing.
100691 In some embodiments, the CRISPR/Cas gene editing is carried out using a
Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b.
100701 In some embodiments, the CRISPR/Cas gene editing is carried out using a
Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
7
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d)
optionally Csfl,
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected
from the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d
100711 In some embodiments, the CRISPR/Cas gene editing is carried out ex vivo
from a
donor subject.
100721 In some embodiments, the CRISPR/Cas gene editing is carried out using a
lentiviral
vector.
100731 In some embodiments, the cell or the progeny thereof evades NK cell
mediated
cytotoxicity upon administration to a patient.
100741 In some embodiments, the cell or the progeny thereof is protected from
cell lysis by
mature NK cells upon administration to a patient.
100751 In some embodiments, the cell or the progeny thereof evades macrophage
engulfment
upon administration to a patient
100761 In some embodiments, the cell or the progeny thereof does not induce an
immune
response to the cell upon administration to a patient.
100771 In some embodiments, the cell or the progeny thereof does not induce an
antibody-
based immune response to the cell upon administration to a patient.
100781 In some embodiments, the wild-type cell or the control cell is a
starting material.
100791 In some embodiments, provided herein is a pharmaceutical composition
comprising a
population of the engineered cells, hypoimmunogenic T cells, non-activated T
cells, pancreatic
islet cells, cardiac muscle cells, glial progenitor cell, or NK cells
described herein, and a
pharmaceutically acceptable additive, carrier, diluent, or excipient.
100801 In some embodiments, the composition comprises one or more populations
of cells
selected from the group consisting of a population of hypoimmunogenic T cells,
a population of
non-activated T cells, a population hypoimmunogenic CD19 CAR T cells, and a
population of
hypoimmunogenic CD22 CAR T cells, and a pharmaceutically acceptable additive,
carrier,
diluent or excipient.
8
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[0081] In some embodiments, provided herein is a method of treating a patient
with a disease
or condition who would benefit from a cell-based therapy, comprising
administering a
population of the engineered cells, hypoimmunogenic T cells, non-activated T
cells, pancreatic
islet cells, cardiac muscle cells, glial progenitor cell, or NK cells
described herein to the patient
[0082] In some embodiments, the patient does not have a Y chromosome.
[0083] In some embodiments, the patient is not sensitized to the Y chromosome
gene
[0084] In some embodiments, the patient is sensitized to the Y chromosome
gene.
[0085] In some embodiments, the patient previously received cell therapy
derived from a
donor subject having a Y chromosome or a cell therapy that otherwise expressed
one or more of
the Y chromosome genes.
[0086] In some embodiments, the patient is a female patient who was previously
pregnant with
a male child.
100871 In some embodiments, provided herein is a method of treating cancer in
a patient in
need thereof comprising administering a population of the primary immune cells
disclosed herein
to the patient.
[0088] In some embodiments, the primary immune cells are selected from the
group consisting
of T cells and NK cells.
[0089] In some embodiments, the patient does not have a Y chromosome.
[0090] In some embodiments, the patient is not sensitized to the Y chromosome
gene.
[0091] In some embodiments, the patient is sensitized to the Y chromosome
gene.
[0092] In some embodiments, the patient previously received cell therapy
derived from a
donor subject having a Y chromosome or a cell therapy that otherwise expressed
one or more of
the Y chromosome genes.
[0093] In some embodiments, the patient is a female patient who was previously
pregnant with
a male child
[0094] In some embodiments, provided herein is a method of determining the
appropriate cell-
based therapy to administer to a patient with a disease or condition who would
benefit from a
9
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
cell-based therapy, comprising: (a) determining whether a biological sample
from the patient
comprises antibodies against one or more Y chromosome genes by: (i) obtaining
or having
obtained a biological sample from the patient; (ii) performing or having
performed an assay to
determine whether antibodies against Protocadherin-1 1 Y-linked are present in
the biological
sample; and (iii) performing or having performed an assay to determine whether
antibodies
against Neuroligin-4 Y-linked are present in the biological sample; and (b)
administering a
population of the engineered cells, hypoimmunogenic T cells, non-activated T
cells, pancreatic
islet cells, cardiac muscle cells, glial progenitor cell, or NK cells
described herein, wherein: (i) if
antibodies against Protocadherin-11 Y-linked are present in the biological
sample, the population
of cells comprises reduced expression of Protocadherin-11 Y-linked; (ii) if
antibodies against
Neuroligin-4 Y-linked are present in the biological sample, the population of
cells comprises
reduced expression of Neuroligin-4 Y-linked; (iii) if antibodies against
Protocadherin-11 Y-
linked and antibodies against Neuroligin-4 Y-linked are present in the
biological sample, the
population of cells comprises reduced expression of Protocadherin-11 Y-linked
and of
Neuroligin-4 Y-linked; (iv) if neither antibodies against Protocadherin-11 Y-
linked nor
antibodies against Neuroligin-4 Y-linked are present in the biological sample,
the population of
cells does not comprise reduced expression of Protocadherin-11 Y-linked or of
Neuroligin-4 Y-
linked.
100951 In some embodiments, provided herein is a method of identifying a
patient with a
disease or condition who would benefit from a cell-based therapy comprising
reduced expression
of one or more Y chromosome genes, the method comprising: (a) determining
whether a
biological sample from the patient comprises antibodies against one or more Y
chromosome
genes by: (i) obtaining or having obtained a biological sample from the
patient; (ii) performing
or having performed an assay to determine whether antibodies against
Protocadherin-11 Y-
linked are present in the biological sample; and (iii) performing or having
performed an assay to
determine whether antibodies against Neuroligin-4 Y-linked are present in the
biological sample;
and (b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK cells
described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked are
present in the biological sample, the population of cells comprises reduced
expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadherin-11 Y-linked and antibodies
against Neuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadherin-11 Y-linked and of Neuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
100961 In some embodiments, provided herein is a method for identifying a
patient with a
disease or condition who would benefit from a cell-based therapy comprising
reduced expression
of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-linked, the method
comprising: (a)
determining whether a biological sample from the patient comprises antibodies
against
Protocadherin-1 1 Y-linked and/or antibodies against Neuroligin-4 Y-linked by:
(i) obtaining or
having obtained a biological sample from the patient; (ii) performing or
having performed an
assay to determine whether antibodies against Protocadherin-11 Y-linked are
present in the
biological sample; and (iii) performing or having performed an assay to
determine whether
antibodies against Neuroligin-4 Y-linked are present in the biological sample;
and (b)
administering a population of the engineered cells, hypoimmunogenic T cells,
non-activated T
cells, pancreatic islet cells, cardiac muscle cells, glial progenitor cell, or
NK cells described
herein to the patient, wherein: (i) if antibodies against Protocadherin-11 Y-
linked are present in
the biological sample, the population of cells comprises reduced expression of
Protocadherin-11
Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked are present in the
biological sample, the
population of cells comprises reduced expression of Neuroligin-4 Y-linked;
(iii) if antibodies
against Protocadherin-11 Y-linked and antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells comprises reduced expression of
Protocadherin-11
Y-linked and of Neuroligin-4 Y-linked; (iv) if neither antibodies against
Protocadherin-11 Y-
linked nor antibodies against Neuroligin-4 Y-linked are present in the
biological sample, the
population of cells does not comprise reduced expression of Protocadherin-1 1
Y-linked or of
Neuroligin-4 Y-linked.
100971 In some embodiments, provided herein is a method of determining whether
a cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
11
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
Neuroligin-4 Y-linked is susceptible to NK mediated cytotoxicity upon
administration to a
patient, the method comprising: (a) determining whether a biological sample
from the patient
comprises antibodies against Protocadherin-11 Y-linked and/or antibodies
against Neuroligin-4
Y-linked by: (i) obtaining or having obtained a biological sample from the
patient; (ii)
performing or having performed an assay to determine whether antibodies
against Protocadherin-
11 Y-linked are present in the biological sample; and (iii) performing or
having performed an
assay to determine whether antibodies against Neuroligin-4 Y-linked are
present in the biological
sample; and (b) administering a population of the engineered cells,
hypoimmunogenic T cells,
non-activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked
are present in the biological sample, the population of cells comprises
reduced expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadherin-11 Y-linked and antibodies
against Neuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadhcrin-11 Y-linked and of Ncuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked
100981 In some embodiments, provided herein is a method of determining whether
a cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
Neuroligin-4 Y-linked is susceptible to lysis by mature NK cells upon
administration to a patient,
the method comprising: (a) determining whether a biological sample from the
patient comprises
antibodies against Protocadherin-11 Y-linked and/or antibodies against
Neuroligin-4 Y-linked
by: (i) obtaining or having obtained a biological sample from the patient;
(ii) performing or
having performed an assay to determine whether antibodies against
Protocadherin-11 Y-linked
are present in the biological sample; and (iii) performing or having performed
an assay to
determine whether antibodies against Neuroligin-4 Y-linked are present in the
biological sample;
and (b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK cells
described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked are
12
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
present in the biological sample, the population of cells comprises reduced
expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadherin-11 Y-linked and antibodies
against Neuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadherin-11 Y-linked and of Neuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Newoligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
100991 In some embodiments, provided herein is a method of determining whether
a cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
Neuroligin-4 Y-linked is susceptible to macrophage engulfment upon
administration to a patient,
the method comprising: (a) determining whether a biological sample from the
patient comprises
antibodies against Protocadherin-11 Y-linked and/or antibodies against
Neuroligin-4 Y-linked
by: (i) obtaining or having obtained a biological sample from the patient;
(ii) performing or
having performed an assay to determine whether antibodies against
Protocadherin-11 Y-linked
are present in the biological sample; and (iii) performing or having performed
an assay to
determine whether antibodies against Neuroligin-4 Y-linked are present in the
biological sample;
and (b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK cells
described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked are
present in the biological sample, the population of cells comprises reduced
expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadherin-11 Y-linked and antibodies
against Neuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadherin-11 Y-linked and of Neuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
13
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1001001 In some embodiments, provided herein is a method of determining
whether a cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
Neuroligin-4 Y-linked is susceptible to an induced immune response upon
administration to a
patient, the method comprising: (a) determining whether a biological sample
from the patient
comprises antibodies against Protocadherin-11 Y-linked and/or antibodies
against Neuroligin-4
Y-linked by: (i) obtaining or having obtained a biological sample from the
patient; (ii)
performing or having performed an assay to determine whether antibodies
against Protocadherin-
11 Y-linked are present in the biological sample; and (iii) performing or
having performed an
assay to determine whether antibodies against Neuroligin-4 Y-linked are
present in the biological
sample; and (b) administering a population of the engineered cells,
hypoimmunogenic T cells,
non-activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK
cells described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked
are present in the biological sample, the population of cells comprises
reduced expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadhcrin-11 Y-linked and antibodies
against Ncuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadherin-11 Y-linked and of Neuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
1001011 In some embodiments, provided herein is a method of determining
whether a cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
Neuroligin-4 Y-linked is susceptible to an induced antibody-based immune
response upon
administration to a patient, the method comprising: (a) determining whether a
biological sample
from the patient comprises antibodies against Protocadherin-11 Y-linked and/or
antibodies
against Neuroligin-4 Y-linked by: (i) obtaining or having obtained a
biological sample from the
patient; (ii) performing or having performed an assay to determine whether
antibodies against
Protocadherin-11 Y-linked are present in the biological sample; and (iii)
performing or having
performed an assay to determine whether antibodies against Neuroligin-4 Y-
linked are present in
the biological sample; and (b) administering a population of the engineered
cells,
14
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
hypoimmunogenic T cells, non-activated T cells, pancreatic islet cells,
cardiac muscle cells, glial
progenitor cell, or NK cells described herein to the patient, wherein: (i) if
antibodies against
Protocadherin-11 Y-linked are present in the biological sample, the population
of cells comprises
reduced expression of Protocadherin-1 1 Y-linked; (ii) if antibodies against
Neuroligin-4 Y-
linked are present in the biological sample, the population of cells comprises
reduced expression
of Neuroligin-4 Y-linked; (iii) if antibodies against Protocadherin-11 Y-
linked and antibodies
against Neuroligin-4 Y-linked are present in the biological sample, the
population of cells
comprises reduced expression of Protocadherin-11 Y-linked and of Neuroligin-4
Y-linked; (iv)
if neither antibodies against Protocadherin-11 Y-linked nor antibodies against
Neuroligin-4 Y-
linked are present in the biological sample, the population of cells does not
comprise reduced
expression of Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
1001021 In some embodiments, provided herein is a method of treating a patient
with a disease
or condition who would benefit from a cell-based therapy, comprising: (a)
determining whether a
biological sample from the patient comprises antibodies against one or more Y
chromosome
genes by: (i) obtaining or having obtained a biological sample from the
patient; (ii) performing
or having performed an assay to determine whether antibodies against
Protocadherin-11 Y-
linked are present in the biological sample; and (iii) performing or having
performed an assay to
determine whether antibodies against Neuroligin-4 Y-linked are present in the
biological sample;
and (b) administering a population of the engineered cells, hypoimmunogenic T
cells, non-
activated T cells, pancreatic islet cells, cardiac muscle cells, glial
progenitor cell, or NK cells
described herein to the patient, wherein: (i) if antibodies against
Protocadherin-11 Y-linked are
present in the biological sample, the population of cells comprises reduced
expression of
Protocadherin-11 Y-linked; (ii) if antibodies against Neuroligin-4 Y-linked
are present in the
biological sample, the population of cells comprises reduced expression of
Neuroligin-4 Y-
linked; (iii) if antibodies against Protocadherin-11 Y-linked and antibodies
against Neuroligin-4
Y-linked are present in the biological sample, the population of cells
comprises reduced
expression of Protocadherin-1 1 Y-linked and of Neuroligin-4 Y-linked; (iv) if
neither antibodies
against Protocadherin-11 Y-linked nor antibodies against Neuroligin-4 Y-linked
are present in
the biological sample, the population of cells does not comprise reduced
expression of
Protocadherin-11 Y-linked or of Neuroligin-4 Y-linked.
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00103] In some embodiments, the Y chromosome gene is a Y chromosome linked
antigen or a
minor histocompatibility antigen associated with the Y chromosome.
1001041 In some embodiments, the one or more Y chromosome linked antigens are
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
[00105] In some embodiments, the cell has reduced expression of Protocadherin-
1 1 Y-linked.
[00106] In some embodiments, the cell has reduced expression of Neuroligin-4 Y-
linked.
[00107] In some embodiments, the cell has reduced expression of Protocadherin-
11 Y-linked
and reduced expression of Neuroligin-4 Y-linked.
1001081 In some embodiments, the cell is genetically engineered to have
reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
[00109] In some embodiments, the cell does not express Protocadherin-11 Y-
linked.
[00110] In some embodiments, the cell does not express Neuroligin-4 Y-linked.
[00111] In some embodiments, the cell does not express Protocadherin-11 Y-
linked and does
not express Neuroligin-4 Y-linked.
1001121 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
1 1 Y-linked and/or Neuroligin-4 Y-linked.
[00113] In some embodiments, reduced expression of Protocadherin-11 Y-linked
and/or
Neuroligin-4 Y-linked is caused by a knock out of the PCDH11Y and/or NI_GN4Y
gene,
respectively.
1001141 In some embodiments, the cell is derived from a human cell or an
animal cell.
1001151 In some embodiments, the human cell or animal cell is from a donor
subject that does
not have a Y chromosome.
1001161 In some embodiments, the human cell or animal cell is from a donor
subject that has a
Y chromosome, and the cell is genetically engineered to have reduced
expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
[00117] In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked.
16
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00118] In some embodiments, the cell is genetically engineered to not express
Neuroligin-4 Y-
linked.
1001191 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked and to not express Neuroligin-4 Y-linked.
[00120] In some embodiments, the cell is propagated or derived from a pool of
cells that are
isolated from one or more donor subjects different from the patient, and the
one or more donor
subjects optionally comprise one or more subjects that have a Y chromosome;
one or more
subjects that do not have a Y chromosome; or a mixture of subjects that do
have a Y
chromosome and subjects that do not have a Y chromosome.
[00121] In some embodiments, the cell is genetically engineered to have
reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked using CRISPR/Cas gene
editing.
1001221 In some embodiments, the CRISPR/Cas gene editing is carried out using
one or more
guide RNAs comprising any of the sequences of Tables 2-5.
[00123] In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b.
[00124] In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, Casl Od, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d)
optionally Csfl;
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected
from the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.
[00125] In some embodiments, the CRISPR/Cas gene editing is carried out ex
vivo from a
donor subject.
[00126] In some embodiments, the CRISPR/Cas gene editing is carried out using
a lentiviral
vector.
17
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1001271 In some embodiments, the cell comprises reduced expression of B2M
and/or CIITA
relative to an unaltered or unmodified wild-type or control cell.
1001281 In some embodiments, the cell does not express B2M and/or CIITA.
1001291 In some embodiments, the cell comprises reduced expression of RHD.
1001301 In some embodiments, the cell does not express RHD.
1001311 In some embodiments, the cell is a differentiated cell derived from an
induced
pluripotent stem cell or a progeny thereof.
1001321 In some embodiments, the differentiated cell is selected from the
group consisting of a
T cell, a NK cell, an endothelial cell, a pancreatic islet cell, a cardiac
muscle cell, a smooth
muscle cell, a skeletal muscle cell, a hepatocyte, a glial progenitor cell, a
dopaminergic neuron, a
retinal pigment epithelial cell, and a thyroid cell.
1001331 In some embodiments, the cell is a primary immune cell or a progeny
thereof.
1001341 In some embodiments, the primary immune cell or a progeny thereof is a
T cell or an
NK cell.
1001351 In some embodiments, the cell comprises reduced expression of TCR-
alpha and/or
TCR-beta.
1001361 In some embodiments, the cell does not express TCR-alpha and/or TCR-
beta
1001371 In some embodiments, the cell further comprises a second exogenous
polynucleotide
encoding one or more CARs, and the one or more CARs comprise an extracellular
ligand-
binding domain having specificity for CD19, CD20, CD22, or BCMA, a hinge
domain, a
transmembrane domain, a co-stimulatory domain, and an intracellular signaling
domain.
1001381 In some embodiments, the one or more CARs comprise a CD8a hinge
domain, a CD28
hinge domain, or an IgG4 hinge domain.
1001391 In some embodiments, the one or more CARs comprise a CD8a hinge domain
having
the amino acid sequence of SEQ ID NO: 9.
1001401 In some embodiments, the one or more CARs comprise a CD28 hinge domain
having
the amino acid sequence of SEQ ID NO: 10 or 113.
18
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00141] In some embodiments, the one or more CARs comprise a IgG4 hinge domain
having
the amino acid sequence of SEQ ID NO: 11 or 12.
1001421 In some embodiments, the one or more CARs comprise a CD8a
transmembrane
domain or a CD28 transmembrane domain.
[00143] In some embodiments, the one or more CARs comprise a CD8a
transmembrane
domain having the amino acid sequence of SEQ ID NO: 14.
[00144] In some embodiments, the one or more CARs comprise a CD28
transmembrane domain
having the amino acid sequence of SEQ ID NO: 15 or 114.
[00145] In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain,
a CD28 costimulatory domain, or a CD3t signaling domain.
[00146] In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain
having the amino acid sequence of SEQ ID NO: 16.
1001471 In some embodiments, the one or more CARs comprise a CD28
costimulatory domain
having the amino acid sequence of SEQ ID NO: 17.
[00148] In some embodiments, the one or more CARs comprise a CD3C signaling
domain
having the amino acid sequence of SEQ ID NO: 18 or 115,
[00149] In some embodiments, the one or more CARs comprise an extracellular
ligand-binding
domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54,
63, 72, 81, or
118, or the CARs have an scFy sequence comprising the heavy and light chain
sequences of any
one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.
[00150] In some embodiments, the one or more CARs have a sequence of any one
of SEQ ID
NOs: 32, 34, 36, 117, or 128.
[00151] In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:117 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:117, with
the following
components: CD8a signal peptide, FMC63 scFy (VL-Whitlow linker-VH), CD8a,
hinge domain,
CD8a transmembrane domain, 4-BB costimulatory domain, and CD3 signaling
domain.
19
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1001521 In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:45 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:45.
1001531 In some embodiments, one or more of the first and/or second exogenous
polynucleotides is inserted into a first and/or second specific locus of at
least one allele of the
cell.
1001541 In some embodiments, the first and/or second specific loci are
selected from the group
consisting of a safe harbor or target locus, an RHD locus, a B2M locus, a
CIITA locus, a TRAC
locus, and a TRB locus.
1001551 In some embodiments, the safe harbor or target locus is selected from
the group
consisting of a CCR5 locus, a CXCR./ locus, a
1 R12C locus, an ALB locus, a SHS'231 locus,
a CIYBI locus, a Rosa locus, an F3 (CD 142) locus, a MICA locus, a MICR locus,
a 1RP 1
(CD91) locus, a HMGB 1 locus, an ABO locus, a FUT 1 locus, and a KDM5D locus.
1001561 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the cells using a gene therapy vector or a transposase system selected
from the group
consisting of transposases, PiggyBac transposons, Sleeping Beauty (SB11)
transposons, Mosl
transposons, and To12 transposons.
1001571 In some embodiments, the gene therapy vector is a retrovirus or a
fusosome.
1001581 In some embodiments, the retrovirus is a lentiviral vector.
1001591 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the cell using CRISPR/Cas gene editing.
1001601 In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b.
1001611 In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d)
optionally Csfl,
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (1) optionally selected
from the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d
1001621 In some embodiments, the CRISPR/Cas gene editing is carried out ex
vivo from a
donor subject.
1001631 In some embodiments, the CRISPR/Cas gene editing is carried out using
a lentiviral
vector.
1001641 In some embodiments, the cell or the progeny thereof evades NK cell
mediated
cytotoxicity upon administration to a patient.
1001651 In some embodiments, the cell or the progeny thereof is protected from
cell lysis by
mature NK cells upon administration to a patient.
1001661 In some embodiments, the cell or the progeny thereof evades macrophage
engulfment
upon administration to a patient
1001671 In some embodiments, the cell or the progeny thereof does not induce
an immune
response to the cell upon administration to a patient.
1001681 In some embodiments, the cell or the progeny thereof does not induce
an antibody-
based immune response to the cell upon administration to a patient.
1001691 In some embodiments, the wild-type cell or the control cell is a
starting material.
1001701 In some embodiments, provided herein is a use of a population of
engineered T cells for
treating a disorder or condition in a patient, wherein the engineered T cells
comprise reduced
expression of one or more Y chromosome genes and MHC class I and/or class II
human
leukocyte antigen molecules relative to an unaltered or unmodified wild-type
or control cell, and
a first exogenous polynucleotide encoding CD47, wherein the engineered T cells
are propagated
from a primary T cell or a progeny thereof, or are derived from an iPSC or a
progeny thereof.
1001711 In some embodiments, provided herein is a use of a population of
engineered
differentiated cells for treating a disorder or condition in a patient,
wherein the engineered
differentiated cells comprise reduced expression of one or more Y chromosome
genes and MEC
21
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
class I and/or class II human leukocyte antigen molecules relative to an
unaltered or unmodified
wild-type or control cell, and a first exogenous polynucleotide encoding CD47,
wherein the
engineered differentiated cells are derived an iPSC or a progeny thereof
1001721 In some embodiments, the Y chromosome gene is a Y chromosome linked
antigen or a
minor histocompatibility antigen associated with the Y chromosome.
1001731 In some embodiments, the one or more Y chromosome linked antigens are
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
1001741 In some embodiments, the cell has reduced expression of Protocadherin-
11 Y-linked.
1001751 In some embodiments, the cell has reduced expression of Neuroligin-4 Y-
linked.
1001761 In some embodiments, the cell has reduced expression of Protocadherin-
11 Y-linked
and reduced expression of Neuroligin-4 Y-linked.
1001771 In some embodiments, the cell is genetically engineered to have
reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
1001781 In some embodiments, the cell does not express Protocadherin-11 Y-
linked
1001791 In some embodiments, the cell does not express Neuroligin-4 Y-linked.
1001801 In some embodiments, the cell does not express Protocadherin-11 Y-
linked and does
not express Neuroligin-4 Y-linked.
1001811 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked and/or Neuroligin-4 Y-linked.
1001821 In some embodiments, reduced expression of Protocadherin-11 Y-linked
and/or
Neuroligin-4 Y-linked is caused by a knock out of the PCDH11Y and/or NI_GN4Y
gene,
respectively.
1001831 In some embodiments, the cell is derived from a human cell or an
animal cell.
1001841 In some embodiments, the human cell or animal cell is from a donor
subject that does
not have a Y chromosome.
22
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1001851 In some embodiments, the human cell or animal cell is from a donor
subject that has a
Y chromosome, and the cell is genetically engineered to have reduced
expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked.
1001861 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked.
1001871 In some embodiments, the cell is genetically engineered to not express
Neuroligin-4 Y-
linked.
1001881 In some embodiments, the cell is genetically engineered to not express
Protocadherin-
11 Y-linked and to not express Neuroligin-4 Y-linked.
1001891 In some embodiments, the cell is propagated or derived from a pool of
cells that are
isolated from one or more donor subjects different from the patient, and the
one or more donor
subjects optionally comprise one or more subjects that have a Y chromosome;
one or more
subjects that do not have a Y chromosome; or a mixture of subjects that do
have a Y
chromosome and subjects that do not have a Y chromosome.
1001901 In some embodiments, the cell is genetically engineered to have
reduced expression of
Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked using CRISPR/Cas gene
editing.
1001911 In some embodiments, the CRISPR/Cas gene editing is carried out using
one or more
guide RNAs comprising any of the sequences of Tables 2-5
1001921 In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b.
1001931 In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10, (d)
optionally Csfl,
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (f) optionally selected
from the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.
23
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00194] In some embodiments, the CRISPR/Cas gene editing is carried out ex
vivo from a
donor subject.
1001951 In some embodiments, the CRISPR/Cas gene editing is carried out using
a lentiviral
vector.
[00196] In some embodiments, the cell comprises reduced expression of B2M
and/or CIITA
relative to an unaltered or unmodified wild-type or control cell.
[00197] In some embodiments, the cell does not express B2M and/or CIITA.
[00198] In some embodiments, the cell comprises reduced expression of RHD.
[00199] In some embodiments, the cell does not express RHD.
[00200] In some embodiments, the cell is a differentiated cell derived from an
induced
pluripotent stem cell or a progeny thereof
[00201] In some embodiments, the differentiated cell is selected from the
group consisting of a
T cell, a NK cell, an endothelial cell, a pancreatic islet cell, a cardiac
muscle cell, a smooth
muscle cell, a skeletal muscle cell, a hepatocyte, a glial progenitor cell, a
dopaminergic neuron, a
retinal pigment epithelial cell, and a thyroid cell.
[00202] In some embodiments, the cell is a primary immune cell or a progeny
thereof.
[00203] In some embodiments, the primary immune cell or a progeny thereof is a
T cell or an
NK cell
[00204] In some embodiments, the cell comprises reduced expression of TCR-
alpha and/or
TCR-beta.
[00205] In some embodiments, the cell does not express TCR-alpha and/or TCR-
beta.
[00206] In some embodiments, the cell further comprises a second exogenous
polynucleotide
encoding one or more CARs, and the one or more CARs comprise an extracellular
ligand-
binding domain having specificity for CD19, CD20, CD22, or BCMA, a hinge
domain, a
transmembrane domain, a co-stimulatory domain, and an intracellular signaling
domain.
[00207] In some embodiments, the one or more CARs comprise a CD8a hinge
domain, a CD28
hinge domain, or an IgG4 hinge domain.
24
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00208] In some embodiments, the one or more CARs comprise a CD8ct hinge
domain having
the amino acid sequence of SEQ ID NO: 9.
1002091 In some embodiments, the one or more CARs comprise a CD28 hinge domain
having
the amino acid sequence of SEQ ID NO: 10 or 113.
[00210] In some embodiments, the one or more CARs comprise a IgG4 hinge domain
having
the amino acid sequence of SEQ ID NO: 11 or 12.
[00211] In some embodiments, the one or more CARs comprise a CD8ct
transmembrane
domain or a CD28 transmembrane domain.
[00212] In some embodiments, the one or more CARs comprise a CD8ct
transmembrane
domain having the amino acid sequence of SEQ ID NO: 14.
[00213] In some embodiments, the one or more CARs comprise a CD28
transmembrane domain
having the amino acid sequence of SEQ ID NO: 15 or 114.
1002141 In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain,
a CD28 costimulatory domain, or a CD3t signaling domain.
[00215] In some embodiments, the one or more CARs comprise a 4-1BB
costimulatory domain
having the amino acid sequence of SEQ ID NO: 16.
[00216] In some embodiments, the one or more CARs comprise a CD28
costimulatory domain
having the amino acid sequence of SEQ ID NO: 17.
[00217] In some embodiments, the one or more CARs comprise a CD3t signaling
domain
having the amino acid sequence of SEQ ID NO: 18 or 115.
[00218] In some embodiments, the one or more CARs comprise an extracellular
ligand-binding
domain comprising an scFv sequence of any one of SEQ ID NOs: 19, 37, 45, 54,
63, 72, 81, or
118, or the CARs have an scEv sequence comprising the heavy and light chain
sequences of any
one of SEQ ID NOs: 20, 25, 38, 42, 46, 50, 64, 68, 73, 77, 119, or 123.
[00219] In some embodiments, the one or more CARs have a sequence of any one
of SEQ ID
NOs: 32, 34, 36, 117, or 128.
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1002201 In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:117 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:117, with
the following
components: CD8a signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD8a hinge
domain,
CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3 signaling
domain.
1002211 In some embodiments, the one or more CARs comprise an amino acid
sequence set
forth in SEQ ID NO:45 or an amino acid sequence at least 80% identical (e.g.,
at least 80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in of SEQ ID NO:45.
1002221 In some embodiments, one or more of the first and/or second exogenous
polynucleotides is inserted into a first and/or second specific locus of at
least one allele of the
cell.
1002231 In some embodiments, the first and/or second specific loci are
selected from the group
consisting of a safe harbor or target locus, an RHD locus, a B2M locus, a
CIITA locus, a TRAC
locus, and a TRB locus.
1002241 In some embodiments, the safe harbor or target locus is selected from
the group
consisting of a CCR5 locus, a CXCR4 locus, a PPP IRI 2C locus, an ALB locus, a
SHS23I locus,
a CLYBL locus, a Rosa locus, an F3 (CD /42) locus, a IfICA locus, a MICB
locus, a LRP 1
(CD91) locus, a 1-JMGB/ locus, an ABO locus, a 11111] locus, and a KDM5D
locus.
1002251 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the engineered T cells using a gene therapy vector or a transposase
system selected from the
group consisting of transposases, PiggyBac transposons, Sleeping Beauty (SB11)
transposons,
Mosl transposons, and To12 transposons.
1002261 In some embodiments, the gene therapy vector is a retrovirus or a
fusosome.
1002271 In some embodiments, the retrovirus is a lentiviral vector.
1002281 In some embodiments, the first and/or second exogenous polynucleotide
is introduced
into the cell using CRISPR/Cas gene editing.
26
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00229] In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of Cas9, Cas12a, and Cas12b
1002301 In some embodiments, the CRISPR/Cas gene editing is carried out using
a Cas effector
protein selected from the group consisting of: (a) optionally selected from
the group consisting of
Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and
GSU0054; (b)
optionally selected from the group consisting of Cas9, Csn2, and Cas4; (c)
optionally selected
from the group consisting of Cas10, Csm2, Cmr5, Cas10, Csx11, and Csx10; (d)
optionally Csfl,
(e) optionally selected from the group consisting of Cas12a, Cas12b, Cas12c,
C2c4, C2c8, C2c5,
C2c10, C2c9, CasX (Cas12e), and CasY (Cas12d); and (0 optionally selected from
the group
consisting of Cas13, Cas13a, C2c2, Cas13b, Cas13c, and Cas13d.
1002311 In some embodiments, the CRISPR/Cas gene editing is carried out ex
vivo from a
donor subject.
[00232] In some embodiments, the CRISPR/Cas gene editing is carried out using
a lentiviral
vector.
1002331 In some embodiments, the cell or the progeny thereof evades NK cell
mediated
cytotoxicity upon administration to a patient.
[00234] In some embodiments, the cell or the progeny thereof is protected from
cell lysis by
mature NK cells upon administration to a patient.
[00235] In some embodiments, the cell or the progeny thereof evades macrophage
engulfment
upon administration to a patient.
1002361 In some embodiments, the cell or the progeny thereof does not induce
an immune
response to the cell upon administration to a patient.
[00237] In some embodiments, the cell or the progeny thereof does not induce
an antibody-
based immune response to the cell upon administration to a patient
[00238] In some embodiments, the wild-type cell or the control cell is a
starting material.
[00239] In some embodiments, provided herein is a method for producing an
engineered cell
comprising reduced expression of one or more Y chromosome genes and MHC class
I and/or
class II human leukocyte antigen molecules relative to an unaltered or
unmodified wild-type or
27
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
control cell, and a first exogenous polynucleotide encoding CD47, the method
comprising: (a)
obtaining an isolated cell; (b) genetically modifying the cell to reduce
expression of the one or
more Y chromosome genes in the cell; (c) genetically modifying the cell to
reduce expression of
MHC class I human leukocyte antigen molecules and/or MHC class IT human
leukocyte antigen
molecules in the cell; and (d) introducing into the isolated cell a
polynucleotide encoding CD47,
to thereby produce the engineered cell.
[00240] In some embodiments, provided herein is a method for producing an
engineered cell
comprising reduced expression of one or more Y chromosome genes and MHC class
I and/or
class II human leukocyte antigen molecules relative to an unaltered or
unmodified wild-type or
control cell, and a first exogenous polynucleotide encoding CD47, the method
comprising: (a)
obtaining an isolated cell; (b) contacting the cell with a composition
comprising lentiviral vectors
comprising (i) a CD4 binding agent or a CD8 binding agent, (ii)
polynucleotides encoding
CRISPR/Cas gene editing components targeting the one or more Y chromosome gene
loci, (iii)
polynucleotides encoding CRISPR/Cas gene editing components targeting the MEC
class I
and/or class II human leukocyte antigen gene loci, and (iv) a first exogenous
polynucleotide
encoding CD47, to thereby produce the engineered cell.
[00241] Detailed descriptions of hypoimmunogenic cells, methods of producing
thereof, and
methods of using thereof are found in W02016183041 filed May 9, 2015;
W02018132783 filed
January 14, 2018; W02018176390 filed March 20, 2018; W02020018615 filed July
17, 2019;
W02020018620 filed July 17, 2019; PCT/US2020/44635 filed July 31, 2020;
W02021022223
filed July 31, 2020; W02021041316 filed August 24, 2020; W02021222285 filed
April 27,
2021, 2020; and W02021222285 filed April 27, 2021, the disclosures including
the examples,
sequence listings and figures are incorporated herein by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[00242] FIG. 1A depicts flow cytometry data measuring Protocadherin-Y and
Neuroligin-Y
levels on the cell surface of iPSCs derived from male donors, compared to
isotype control.
[00243] FIG. 1B depicts flow cytometry data measuring Protocadherin-Y and
Neuroligin-Y
levels on the cell surface of iPSCs derived from female donors, compared to
isotype control.
28
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00244] FIG. 2A depicts flow cytometry data measuring Protocadherin-Y and
Neuroligin-Y
levels on the cell surface of CD3+ T cells from three male donors with blood
type 0 analyzed
after thawing, compared to isotype control.
[00245] FIG. 2B depicts flow cytometry data measuring Protocadherin-Y and
Neuroligin-Y
levels on the cell surface of CD3+ T cells from two male donors with blood
type A analyzed
after thawing, compared to isotype control.
[00246] FIG. 2C depicts flow cytometry data measuring Protocadherin-Y and
Neuroligin-Y
levels on the cell surface of CD3+ T cells from two female donors analyzed
after thawing,
compared to isotype control.
1002471 FIGs. 3A, 3B, and 3C show CDC for HIP T cells from a male donor with
blood type 0
using serum from different volunteers.
[00248] FIGs. 4A, 4B, and 4C show ADCC (NK cells) for HIP T cells from a male
donor with
blood type 0 using serum from different volunteers.
1002491 FIGs. 5A, 5B, and 5C show CDC and ADCC (NK cells) for HIP T cells from
a male
donor with blood type 0 using serum from different volunteers and flow
analysis.
[00250] Other objects, advantages and embodiments of the present disclosure
will be apparent
from the detailed description following.
DETAILED DESCRIPTION
I. INTRODUCTION
[00251] Described herein are engineered or modified immune evasive cells
based, in part, on
the hypoimmune editing platform described in W02018132783, and PCT/US21/65157
filed
12/23/2021, each of which is incorporated herein by reference in its entirety,
including but not
limited to human immune evasive cells. To overcome the problem of a subject's
immune
rejection of these primary and/or stein cell-derived transplants, the
inventors have developed and
describe herein hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent
cells, differentiated
cells derived from such, and primary cells) that represent a viable source for
any transplantable
cell type. Such cells are protected from adaptive and/or innate immune
rejection upon
administration to a recipient subject. Advantageously, the cells disclosed
herein are not rejected
by the recipient subject's immune system, regardless of the subject's genetic
make-up, as they are
protected from adaptive and innate immune rejection upon administration to a
recipient subject.
29
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
In some embodiments, the engineered and/or hypoimmunogenic cells do not
express one or more
Y chromosome genes and/or do not express MHC I and/or II antigen molecules
and/or T-cell
receptors. In certain embodiments, the engineered and/or hypoimmunogenic cells
do not express
one or more Y chromosome genes and do not express MT-IC I antigen. In certain
embodiments,
the engineered and/or hypoimmunogenic cells do not express one or more Y
chromosome genes,
do not express MHC I and/or II antigen molecules and/or T-cell receptors, and
overexpress
CD47 proteins. In certain embodiments, the engineered and/or hypoimmunogenic
cells do not
express one or more Y chromosome genes, do not express MHC I and II antigen
molecules
and/or T-cell receptors, and overexpress CD47 proteins. In certain
embodiments, the engineered
and/or hypoimmunogenic cells such as hypoimmunogenic T cells do not express
one or more Y
chromosome genes, do not express MHC I and/or II antigen molecules and/or T-
cell receptors,
overexpress CD47 proteins, and express exogenous CARs. In certain embodiments,
the
engineered and/or hypoimmunogenic cells such as hypoimmunogenic T cells do not
express one
or more Y chromosome genes, do not express MHC I and II antigen molecules
and/or T-cell
receptors, overexpress CD47 proteins, and express exogenous CARs.
[00252] In some embodiments, hypoimmunogenic cells outlined herein are not
subject to an
innate immune cell rejection. In some instances, hypoimmunogenic cells are not
susceptible to
NK cell-mediated lysis. In some instances, hypoimmunogenic cells are not
susceptible to
macrophage engulfment. In some embodiments, hypoimmunogenic cells do not
induce an
immune response. In some embodiments, hypoimmunogenic cells are useful as a
source of
universally compatible cells or tissues (e.g., universal donor cells or
tissues) that are transplanted
into a recipient subject with little to no immunosuppressant agent needed.
Such
hypoimmunogenic cells retain cell-specific characteristics and features upon
transplantation,
including, e.g., pluripotency, as well as being capable of engraftment and
functioning similarly to
a corresponding native cell.
1002531 The technology disclosed herein utilizes expression of tolerogenic
factors and
modulation (e.g., reduction or elimination) of one or more Y chromosome genes,
and optionally
MHC I molecules, MHC II molecules, and/or TCR expression in human cells. In
some
embodiments, genome editing technologies utilizing rare-cutting endonucleases
(e.g., the
CRISPR/Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease
systems)
are also used to reduce or eliminate expression of genes involved in an immune
response (e.g.,
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
by deleting genomic DNA of genes involved in an immune response or by
insertions of genomic
DNA into such genes, such that gene expression is impacted) in the cells. In
some embodiments,
genome editing technologies or other gene modulation technologies are used to
insert tolerance-
inducing (tolerogenic) factors in human cells, rendering the cells and their
progeny (include any
differentiated cells prepared therefrom) able to evade immune recognition upon
engrafting into a
recipient subject. As such, the cells described herein exhibit modulated
expression of one or
more genes and factors that affect one or more Y chromosome genes, MI-IC I
molecules, MI-IC II
molecules, and/or TCR expression and evade the recipient subject's immune
system.
1002541 The genome editing techniques enable double-strand DNA breaks at
desired locus sites.
These controlled double-strand breaks promote homologous recombination at the
specific locus
sites. This process focuses on targeting specific sequences of nucleic acid
molecules, such as
chromosomes, with endonucleases that recognize and bind to the sequences and
induce a double-
stranded break in the nucleic acid molecule. The double-strand break is
repaired either by an
error-prone non-homologous end-joining (NI-IEJ) or by homologous recombination
(FIR).
1002551 Surprisingly, it was found that iPSCs and T cells from male donors
express the Y
chromosome antigens Protocadhcrin-11 Y-linked and Ncuroligin-4 Y-linked. These
surprising
findings suggest that the source of hypoimmunogenic cells, such as
hypoimmunogenic donor T
cells, non-activated T cells, pancreatic islet cells, or cardiac cells should
lack a Y chromosome or
should be genetically modified to reduce expression of Y chromosome antigens
to avoid
detection and elimination by a recipient's adaptive immune system.
1002561 The practice of the numerous embodiments will employ, unless indicated
specifically to
the contrary, conventional methods of chemistry, biochemistry, organic
chemistry, molecular
biology, microbiology, recombinant DNA techniques, genetics, immunology, and
cell biology
that are within the skill of the art, many of which are described below for
the purpose of
illustration. Such techniques are explained fully in the literature. See,
e.g., Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular
Cloning: A
Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular
Biology (John Wiley
and Sons, updated July 2008); Short Protocols in Molecular Biology: A
Compendium of
Methods from Current Protocols in Molecular Biology, Greene Pub. Associates
and Wiley-
Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL
Press, Oxford,
31
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press,
New York,
1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984);
Perbal, A Practical
Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in
Immunology Q. E.
Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds.,
1991); Annual
Review of Immunology; as well as monographs in journals such as Advances in
Immunology.
DEFINITIONS
[00257] As described in the present disclosure, the following terms will be
employed, and are
defined as indicated below.
[00258] The term "antigen", as used herein, refers to a molecule
capable of provoking an
immune response. Antigens include but are not limited to cells, cell extracts,
proteins,
polypeptides, peptides, polysaccharides, polysaccharide conjugates, peptide
and non-peptide
mimics of polysaccharides and other molecules, small molecules, lipids,
glycolipids,
carbohydrates, viruses and viral extracts and multicellular organisms such as
parasites and
allergens. The term antigen broadly includes any type of molecule which is
recognized by a host
immune system as being foreign.
[00259] The terms "autoimmune disease" or "autoimmune disorder" or
"inflammatory disease"
or "inflammatory disorder" refer to any disease or disorder in which the
subject mounts an
immune response against its own tissues and/or cells. Autoimmune disorders can
affect almost
every organ system in the subject (e.g., human), including, but not limited
to, diseases of the
nervous, gastrointestinal, and endocrine systems, as well as skin and other
connective tissues,
eyes, blood and blood vessels. Examples of autoimmune diseases include, but
are not limited to
Hashimoto's thyroiditis, Systemic lupus erythematosus, Sjogren's syndrome,
Graves' disease,
Scleroderma, Rheumatoid arthritis, Multiple sclerosis, Myasthenia gravis and
Diabetes.
[00260] The term "cancer" as used herein is defined as a hyperproliferation of
cells whose
unique trait (e.g., loss of normal controls) results in unregulated growth,
lack of differentiation,
local tissue invasion, and metastasis. With respect to the inventive methods,
the cancer can be
any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia,
alveolar
rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer,
cancer of the anus,
anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile
duct, cancer of the
32
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal
cavity, or middle ear,
cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia,
chronic myeloid
cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma,
gastrointestinal carcinoid
tumor, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer,
leukemia, liquid
tumors, liver cancer, lung cancer, lymphoma, malignant mesothelioma,
mastocytoma, melanoma,
multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer,
pancreatic
cancel, peritoneum, omentum, and mesentely cancer, pharynx cancel, prostate
cancer, rectal
cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer,
solid tumors, stomach
cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder
cancer. As used
herein, the term "tumor" refers to an abnormal growth of cells or tissues of
the malignant type,
unless otherwise specifically indicated and does not include a benign type
tissue.
[00261] The term "cell" refers to any human cell or animal cell. In some
embodiments, the cell
is a human cell or an animal cell that is from a donor subject that has a Y
chromosome. In some
embodiments, the cell is a human cell or an animal cell that is from a donor
subject that does not
have a Y chromosome.
[00262] The term "chronic infectious disease" refers to a disease caused by an
infectious agent
wherein the infection has persisted. Such a disease may include hepatitis (A,
B, or C), herpes
virus (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV/AIDS. Non-viral

examples may include chronic fungal diseases such Aspergillosis, Candidiasis,
Coccidioidomycosis, and diseases associated with Cryptococcus and
Histoplasmosis. None
limiting examples of chronic bacterial infectious agents may be Chlamydia
pneumoniae, Listeria
monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the
disorder is human
immunodeficiency virus (HIV) infection. In some embodiments, the disorder is
acquired
immunodeficiency syndrome (AIDS).
1002631 As used herein, "clinically effective amount" refers to an
amount sufficient to
provide a clinical benefit in the treatment and/or management of a disease,
disorder, or condition.
In some embodiments, a clinically effective amount is an amount that has been
shown to produce
at least one improved clinical endpoint to the standard of care for the
disease, disorder, or
condition. In some embodiments, a clinically effective amount is an amount
that has been
demonstrated, for example in a clinical trial, to be sufficient to provide
statistically significant
and meaningful effectiveness for treating the disease, disorder, or condition.
In some
33
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, the clinically effective amount is also a therapeutically
effective amount. In other
embodiments, the clinically effective amount is not a therapeutically
effective amount.
1002641 In some embodiments, an alteration or modification
(including, for example,
genetic alterations or modifications) described herein results in reduced
expression of a target or
selected polynucleotide sequence. In some embodiments, an alteration or
modification described
herein results in reduced expression of a target or selected polypeptide
sequence. In some
embodiments, an alteration or modification described herein results in
increased expression of a
target or selected polynucleotide sequence. In some embodiments, an alteration
or modification
described herein results in increased expression of a target or selected
polypeptide sequence.
1002651 In additional or alternative embodiments, the present
disclosure contemplates
altering target polynucleotide sequences in any manner which is available to
the skilled artisan,
e.g., utilizing a TALEN system or RNA-guided transposases. It should be
understood that
although examples of methods utilizing CRISPR/Cas (e.g., Cas9 and Cas12a) and
TALEN are
described in detail herein, the present disclosure is not limited to the use
of these
methods/systems. Other methods of targeting, e.g., B2M, to reduce or ablate
expression in target
cells known to the skilled artisan can be utilized herein.
1002661 The terms "decrease," "reduced," "reduction," and "decrease" are all
used herein
generally to mean a decrease by a statistically significant amount. However,
for avoidance of
doubt, decrease," "reduced," "reduction," "decrease" means a decrease by at
least 10% as
compared to a reference level, for example a decrease by at least about 20%,
or at least about
30%, or at least about 40%, or at least about 50%, or at least about 60%, or
at least about 70%, or
at least about 80%, or at least about 90% or up to and including a 100%
decrease (i.e. absent
level as compared to a reference sample), or any decrease between 10-100% as
compared to a
reference level. In some embodiments, the cells are engineered to have reduced
expression of
one or more targets relative to an unaltered or unmodified wild-type or
control cell.
1002671 In some embodiments, the engineered and/or hypoimmunogenic cells
described are
derived from an iPSC or a progeny thereof. As used herein, the term "derived
from an iPSC or a
progeny thereof' encompasses the initial iPSC that is generated and any
subsequent progeny
thereof. As used herein, the term "progeny" encompasses, e.g., a first-
generation progeny, i.e.,
the progeny is directly derived from, obtained from, obtainable from or
derivable from the initial
iPSC by, e.g., traditional propagation methods. The term "progeny" also
encompasses further
34
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
generations such as second, third, fourth, fifth, sixth, seventh, or more
generations, i.e.,
generations of cells which are derived from, obtained from, obtainable from or
derivable from
the former generation by, e.g., traditional propagation methods. The term
"progeny" also
encompasses modified cells that result from the modification or alteration of
the initial iPSC or a
progeny thereof.
[00268] The term "donor subject" refers to an animal, for example, a human
from whom cells
can be obtained. The "non-human animals" and "non-human mammals" as used
interchangeably
herein, includes mammals such as rats, mice, rabbits, sheep, cats, dogs, cows,
pigs, and non-
human primates. The term "donor subject" also encompasses any vertebrate
including but not
limited to mammals, reptiles, amphibians and fish. However, advantageously,
the donor subject
is a mammal such as a human, or other mammals such as a domesticated mammal,
e.g. dog, cat,
horse, and the like, or production mammal, e.g. cow, sheep, pig, and the like.
A "donor subject"
can also refere to more than one donor, for example one or more humans or non-
human animals
or non-human mammals.
[00269] The term "endogenous" refers to a referenced molecule or polypeptide
that is naturally
present in the cell. Similarly, the term when used in reference to expression
of an encoding
nucleic acid refers to expression of an encoding nucleic acid naturally
contained within the cell
and not exogenously introduced. Similarly, the term when used in reference to
a promoter
sequence refers to a promoter sequence naturally contained within the cell and
not exogenously
introduced.
[00270] The term -engineered cell" as used herein refers to a cell
that has been altered in at
least some way by human intervention, including, for example, by genetic
alterations or
modifications such that the engineered cell differs from a wild-type cell.
[00271] As used herein, the term "exogenous" in the context of a
polynucleotide or polypeptide
being expressed is intended to mean that the referenced molecule or the
referenced polypeptide is
introduced into the cell of interest. The polypeptide can be introduced, for
example, by
introduction of an encoding nucleic acid into the genetic material of the
cells such as by
integration into a chromosome or as non-chromosomal genetic material such as a
plasmid or
expression vector. Therefore, the term as it is used in reference to
expression of an encoding
nucleic acid refers to introduction of the encoding nucleic acid in an
expressible form into the
cell.
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1002721 A "gene," for the purposes of the present disclosure, includes a DNA
region encoding a
gene product, as well as all DNA regions which regulate the production of the
gene product,
whether or not such regulatory sequences are adjacent to coding and/or
transcribed sequences.
Accordingly, a gene includes, but is not necessarily limited to, promoter
sequences, terminators,
translational regulatory sequences such as ribosome binding sites and internal
ribosome entry
sites, enhancers, silencers, insulators, boundary elements, replication
origins, matrix attachment
sites and locus control legions.
1002731 "Gene expression" refers to the conversion of the information,
contained in a gene, into
a gene product. A gene product can be the direct transcriptional product of a
gene (e.g., mRNA,
tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA)
or a protein
produced by translation of an mRNA. Gene products also include RNAs which are
modified, by
processes such as capping, polyadenylation, methylation, and editing, and
proteins modified by,
for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-
ribosylation,
myristoylation, and/or glycosylation.
1002741
The term "genetic modification" and its grammatical equivalents as used
herein
can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid
within an organism's
genome. For example, genetic modification can refer to alterations, additions,
and/or deletion of
genes or portions of genes or other nucleic acid sequences. A genetically
modified cell can also
refer to a cell with an added, deleted and/or altered gene or portion of a
gene. A genetically
modified cell can also refer to a cell with an added nucleic acid sequence
that is not a gene or
gene portion. Genetic modifications include, for example, both transient knock-
in or knock-down
mechanisms, and mechanisms that result in permanent knock-in, knock-down, or
knock-out of
target genes or portions of genes or nucleic acid sequences Genetic
modifications include, for
example, both transient knock-in and mechanisms that result in permanent knock-
in of nucleic
acids seqeunces Genetic modifications also include, for example, reduced or
increased
transcription, reduced or increased mRNA stability, reduced or increased
translation, and
reduced or increased protein stability.
1002751 As used herein, the terms "grafting", "administering," "introducing",
"implanting" and
"transplanting" as well as grammatical variations thereof are used
interchangeably in the context
of the placement of cells (e.g., cells described herein) into a subject, by a
method or route which
results in localization or at least partial localization of the introduced
cells at a desired site or
36
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
systemic introduction (e.g., into circulation). The cells can be implanted
directly to the desired
site, or alternatively be administered by any appropriate route which results
in delivery to a
desired location in the subject where at least a portion of the implanted
cells or components of
the cells remain viable. The period of viability of the cells after
administration to a subject can be
as short as a few hours, e. g. twenty-four hours, to a few days, to as long as
several years. In
some embodiments, the cells can also be administered (e.g., injected) a
location other than the
desired site, such as in the brain or subcutaneously, for example, in a
capsule to maintain the
implanted cells at the implant location and avoid migration of the implanted
cells.
1002761 By "HLA" or "human leukocyte antigen" or "HLA molecules" or "human
leukocyte
antigen molecules" complex is a gene complex encoding the MHC proteins in
humans. These
cell-surface proteins that make up the HLA complex are responsible for the
regulation of the
immune response to antigens. In humans, there are two MHCs, class I molecules
and class II
molecules, "HLA-I" and "HLA-II", or "HLA-I molecules" and "HLA-II molecules".
HLA-I
includes three proteins, HLA-A, HLA-B and HLA-C, which present peptides from
the inside of
the cell, and antigens presented by the HLA-I complex attract killer T-cells
(also known as CD8+
T-cells or cytotoxic T cells). The HLA-I proteins are associated with 13-2
microglobulin (B2M).
HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR,
which
present antigens from outside the cell to T lymphocytes. This stimulates CD4+
cells (also known
as T-helper cells). It should be understood that the use of either "MT-IC' or
"HLA" is not meant to
be limiting, as it depends on whether the genes are from humans (HLA) or
murine (1VIFIC). Thus,
as it relates to mammalian cells, these terms may be used interchangeably
herein.
1002771 As used herein to characterize a cell, the term "hypoimmunogenic"
generally means
that such cell is less prone to innate or adaptive immune rejection by a
subject into which such
cells are transplanted, e.g., the cell is less prone to allorejection by a
subject into which such cells
are transplanted. For example, relative to a cell of the same cell type that
does not comprise the
modifications, such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%,
30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to innate or
adaptive immune
rejection by a subject into which such cells are transplanted. In some
embodiments, genome
editing technologies are used to modulate the expression of WIC I and WIC II
genes, and thus,
contribute to generation of a hypoimmunogenic cell. In some embodiments, a
hypoimmunogenic cell evades immune rejection in an MI-IC-mismatched allogeneic
recipient.
37
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
In some instance, differentiated cells produced from the hypoimmunogenic stem
cells outlined
herein evade immune rejection when administered (e.g., transplanted or
grafted) to an MHC-
mismatched allogeneic recipient. In some embodiments, a hypoimmunogenic cell
is protected
from T cell-mediated adaptive immune rejection and/or innate immune cell
rejection Detailed
descriptions of hypoimmunogenic cells, methods of producing thereof, and
methods of using
thereof are found in W02016183041 filed May 9, 2015; W02018132783 filed
January 14, 2018;
W02018176390 filed March 20, 2018, W02020018615 filed July 17, 2019,
W02020018620
filed July 17, 2019; PCT/US2020/44635 filed July 31, 2020; W02021022223 filed
July 31,
2020; W02021041316 filed August 24, 2020; W02021222285 filed April 27, 2021,
2020; and
W02021222285 filed April 27, 2021, the disclosures including the examples,
sequence listings
and figures are incorporated herein by reference in their entirety.
1002781 Hypoimmunogenicity of a cell can be determined by evaluating the
immunogenicity of
the cell such as the cell's ability to elicit adaptive and innate immune
responses or to avoid
eliciting such adaptive and innate immune responses. Such immune response can
be measured
using assays recognized by those skilled in the art. In some embodiments, an
immune response
assay measures the effect of a hypoimmunogenic cell on T cell proliferation, T
cell activation, T
cell killing, donor specific antibody generation, NK cell proliferation, NK
cell activation, and
macrophage activity. In some cases, hypoimmunogenic cells and derivatives
thereof undergo
decreased killing by T cells and/or NK cells upon administration to a subject.
In some instances,
the cells and derivatives thereof show decreased macrophage engulfment
compared to an
unmodified or wild-type cell. In some embodiments, a hypoimmunogenic cell
elicits a reduced
or diminished immune response in a recipient subject compared to a
corresponding unmodified
wild-type cell. In some embodiments, a hypoimmunogenic cell is nonimmunogenic
or fails to
elicit an immune response in a recipient subject.
1002791 The term percent "identity," in the context of two or more nucleic
acid or polypeptide
sequences, refers to two or more sequences or subsequences that have a
specified percentage of
nucleotides or amino acid residues that are the same, when compared and
aligned for maximum
correspondence, as measured using one of the sequence comparison algorithms
described below
(e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or
by visual
inspection. Depending on the application, the percent "identity" can exist
over a region of the
sequence being compared, e.g., over a functional domain, or, alternatively,
exist over the full
38
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
length of the two sequences to be compared. For sequence comparison, typically
one sequence
acts as a reference sequence to which test sequences are compared. When using
a sequence
comparison algorithm, test and reference sequences are input into a computer,
subsequence
coordinates are designated, if necessary, and sequence algorithm program
parameters are
designated. The sequence comparison algorithm then calculates the percent
sequence identity for
the test sequence(s) relative to the reference sequence, based on the
designated program
parameters.
1002801 Optimal alignment of sequences for comparison can be conducted, e.g.,
by the local
homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the
homology
alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the
search for
similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444
(11988), by
computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in
the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science
Dr.,
Madison, Wis.), or by visual inspection (see generally Ausubel et al, infra).
1002811 One example of an algorithm that is suitable for determining percent
sequence identity
and sequence similarity is the BLAST algorithm, which is described in Altschul
et al, J. Mol.
Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly
available
through the National Center for Biotechnology Information.
1002821 "Immune signaling factor" as used herein refers to, in some cases, a
molecule, protein,
peptide and the like that activates immune signaling pathways.
1002831 "Immunosuppressive factor" or "immune regulatory factor" or
"tolerogenic factor" as
used herein include hypoimmunity factors, complement inhibitors, and other
factors that
modulate or affect the ability of a cell to be recognized by the immune system
of a host or
recipient subject upon administration, transplantation, or engraftment. These
may be in
combination with additional genetic modifications.
1002841 The terms "increased", "increase" or "enhance" or "activate" are all
used herein to
generally mean an increase by a statically significant amount; for the
avoidance of any doubt, the
terms "increased", "increase" or "enhance" or "activate" means an increase of
at least 10% as
compared to a reference level, for example an increase of at least about 20%,
or at least about
30%, or at least about 40%, or at least about 50%, or at least about 60%, or
at least about 70%, or
at least about 80%, or at least about 90% or up to and including a 100%
increase or any increase
39
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
between 10-100% as compared to a reference level, or at least about a 2-fold,
or at least about a
3-fold, or at least about a 4-fold, or at least about a 5-fold or at least
about a 10-fold increase, or
any increase between 2-fold and 10-fold or greater as compared to a reference
level. In some
embodiments, the reference level, also referred to as the basal level, is 0.
1002851 In some embodiments, the alteration is an indel. As used herein,
"indel" refers to a
mutation resulting from an insertion, deletion, or a combination thereof. As
will be appreciated
by those skilled in the art, an indel in a coding legion of a genomic sequence
will result in a
frameshift mutation, unless the length of the indel is a multiple of three. In
some embodiments,
the alteration is a point mutation. As used herein, "point mutation" refers to
a substitution that
replaces one of the nucleotides. A gene editing (e.g. CRISPR/Cas) system of
the present
disclosure can be used to induce an indel of any length or a point mutation in
a target
polynucleotide sequence.
1002861 As used herein, "knock down" refers to a reduction in
expression of the target
mRNA or the corresponding target protein. Knock down is commonly reported
relative to levels
present following administration or expression of a noncontrol molecule that
does not mediate
reduction in expression levels of RNA (e.g., a non-targeting control shRNA,
siRNA, or miRNA).
In some embodiments, knock down of a target gene is achived by way of
conditional or
inducible shRNAs, conditional or inducible siRNAs, conditional or inducible
miRNAs, or
conditional or inducible CRISPR interference (CRISPRi). In some embodiments,
knock down of
a target gene is achieved by way of a protein-based method, such as a
conditional or inducible
degron method. In some embodiments, knock down of a target gene is achieved by
genetic
modification, including shRNAs, siRNAs, miRNAs, or use of gene editing systems
(e.g.
CRISPR/Cas).
1002871 Knock down is commonly assessed by measuring the mRNA levels using
quantitative
polymerase chain reaction (qPCR) amplification or by measuring protein levels
by western blot
or enzyme-linked immunosorbent assay (ELISA). Analyzing the protein level
provides an
assessment of both mRNA cleavage as well as translation inhibition. Further
techniques for
measuring knock down include RNA solution hybridization, nuclease protection,
northern
hybridization, gene expression monitoring with a microarray, antibody binding,

radioimmunoassay, and fluorescence activated cell analysis. Those skilled in
the art will readily
appreciate how to use the gene editing systems (e.g., CRISPR/Cas) of the
present disclosure to
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
knock out a target polynucleotide sequence or a portion thereof based upon the
details described
herein
1002881 By "knock in" or "knock-in" herein is meant a genetic
modification resulting from
the insertion of a DNA sequence into a chromosomal locus in a host cell. This
causes initiation
of or increased levels of expression of the knocked in gene, portion of gene,
or nucleic acid
sequence inserted product, e.g., an increase in RNA transcript levels and/or
encoded protein
levels. As will be appreciated by those in the art, this can be accomplished
in several ways,
including inserting or adding one or more additional copies of the gene or
portion thereof to the
host cell or altering a regulatory component of the endogenous gene increasing
expression of the
protein is made or inserting a specific nucleic acid sequence whose expression
is desired. This
may be accomplished by modifying a promoter, adding a different promoter,
adding an enhancer,
adding other regulatory elements, or modifying other gene expression
sequences.
1002891 As used herein, "knock out" or "knock-out" includes
deleting all or a portion of a
target polynucleotide sequence in a way that interferes with the translation
or function of the
target polynucleotide sequence. For example, a knock out can be achieved by
altering a target
polynucleotide sequence by inducing an insertion or a deletion ("inder) in the
target
polynucleotide sequence, including in a functional domain of the target
polynucleotide sequence
(e.g., a DNA binding domain). Those skilled in the art will readily appreciate
how to use the
gene editing systems (e.g. CRISPR/Cas) of the present disclosure to knock out
a target
polynucleotide sequence or a portion thereof based upon the details described
herein.
1002901 In some embodiments, a genetic modification or alteration
results in a knock out or
knock down of the target polynucleotide sequence or a portion thereof.
Knocking out a target
polynucleotide sequence or a portion thereof using a gene editing system (e.g.
CRISPR/Cas) of
the present disclosure can be useful for a variety of applications. For
example, knocking out a
target polynucleotide sequence in a cell can be performed in vitro for
research purposes. For ex
vivo purposes, knocking out a target polynucleotide sequence in a cell can be
useful for treating
or preventing a disorder associated with expression of the target
polynucleotide sequence (e.g.,
by knocking out a mutant allele in a cell ex vivo and introducing those cells
comprising the
knocked out mutant allele into a subject) or for changing the genotype or
phenotype of a cell.
1002911 "Modulation" of gene expression refers to a change in the expression
level of a gene.
Modulation of expression can include, but is not limited to, gene activation
and gene repression.
41
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
Modulation may also be complete, i.e. wherein gene expression is totally
inactivated or is
activated to wild-type levels or beyond; or it may be partial, wherein gene
expression is partially
reduced, or partially activated to some fraction of wild-type levels.
1002921
In additional or alternative aspects, the present disclosure contemplates
altering
target polynucleotide sequences in any manner which is available to the
skilled artisan, e.g.,
utilizing a nuclease system such as a TAL effector nuclease (TALEN) or zinc
finger nuclease
(ZFN) system. It should be understood that although examples of methods
utilizing CRISPR/Cas
(e.g., Cas9 and Cas12a) and TALEN are described in detail herein, the
disclosure is not limited
to the use of these methods/systems. Other methods of targeting to reduce or
ablate expression
in target cells known to the skilled artisan can be utilized herein. The
methods provided herein
can be used to alter a target polynucleotide sequence in a cell. The present
disclosure
contemplates altering target polynucleotide sequences in a cell for any
purpose. In some
embodiments, the target polynucleotide sequence in a cell is altered to
produce a mutant cell. As
used herein, a "mutant cell" refers to a cell with a resulting genotype that
differs from its original
genotype. In some instances, a "mutant cell" exhibits a mutant phenotype, for
example when a
normally functioning gene is altered using the gene editing systems (e.g.,
CRISPR/Cas) systems
of the present disclosure. In other instances, a "mutant cell" exhibits a wild-
type phenotype, for
example when a gene editing system (e.g., CRISPR/Cas) system of the present
disclosure is used
to correct a mutant genotype. In some embodiments, the target polynucleotide
sequence in a cell
is altered to correct or repair a genetic mutation (e.g., to restore a normal
phenotype to the cell)
In some embodiments, the target polynucleotide sequence in a cell is altered
to induce a genetic
mutation (e.g., to disrupt the function of a gene or genomic element).
1002931 "Neuroligin-4 Y-linked", "Neuroligin-4-Y", and "NLGN4Y" and variations
thereof
refer to the Y chromosome linked antigen encoded by the NLGN4Y gene.
1002941 The term "operatively linked" or "operably linked" are used
interchangeably with
reference to a juxtaposition of two or more components (such as sequence
elements), in which
the components are arranged such that both components function normally and
allow the
possibility that at least one of the components can mediate a function that is
exerted upon at least
one of the other components. By way of illustration, a transcriptional
regulatory sequence, such
as a promoter, is operatively linked to a coding sequence if the
transcriptional regulatory
sequence controls the level of transcription of the coding sequence in
response to the presence or
42
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
absence of one or more transcriptional regulatory factors. A transcriptional
regulatory sequence
is generally operatively linked in cis with a coding sequence, but need not be
directly adjacent to
it. For example, an enhancer is a transcriptional regulatory sequence that is
operatively linked to
a coding sequence, even though they are not contiguous.
1002951 The term "patient" or "recipient patient" refers to an animal, for
example, a human to
whom treatment, including prophylactic treatment, with the cells as described
herein, is
provided. For treatment of those infections, conditions or disease states,
which are specific for a
specific animal such as a human patient, the term patient refers to that
specific animal. The term
"patient" also encompasses any vertebrate including but not limited to
mammals, reptiles,
amphibians and fish. However, advantageously, the patient is a mammal such as
a human, or
other mammals such as a domesticated mammal, e.g. dog, cat, horse, and the
like, or production
mammal, e.g. cow, sheep, pig, and the like.
1002961 "Pluripotent stem cells" as used herein have the potential
to differentiate into any
of the three germ layers: endoderm (e.g., the stomach linking,
gastrointestinal tract, lungs, etc.),
mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.) or ectoderm
(e.g., epidermal tissues
and nervous system tissues). The term "pluripotent stem cells," as used
herein, also encompasses
"induced pluripotent stem cells", or "iPSCs", or a type of pluripotent stem
cell derived from a
non-pluripotent cell. In some embodiments, a pluripotent stem cell is produced
or generated
from a cell that is not a pluripotent cell. In other words, pluripotent stem
cells can be direct or
indirect progeny of a non-pluripotent cell. Examples of parent cells include
somatic cells that
have been reprogrammed to induce a pluripotent, undifferentiated phenotype by
various means.
Such" iPS" or "iPSC" cells can be created by inducing the expression of
certain regulatory genes
or by the exogenous application of certain proteins. Methods for the induction
of iPS cells are
known in the art and are further described below. (See, e.g., Zhou et at.,
Stem Cells 27 (11):
2667-74 (2009); Huangfu et at., Nature Biotechnol. 26 (7): 795 (2008); Woltj
en et al., Nature
458 (7239): 766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009);
each of which is
incorporated by reference herein in their entirety.) The generation of induced
pluripotent stem
cells (iPSCs) is outlined below. As used herein, "hiPSCs" are human induced
pluripotent stem
cells. In some embodiments, "pluripotent stem cells," as used herein, also
encompasses
mesenchymal stem cells (MSCs), and/or embryonic stem cells (ESCs).
43
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1002971 As used herein, "promoter," "promoter sequence," or
"promoter region" refers to a
DNA regulatory region/sequence capable of binding RNA polymerase and involved
in initiating
transcription of a downstream coding or non-coding sequence. In some examples,
the promoter
sequence includes the transcription initiation site and extends upstream to
include the minimum
number of bases or elements necessary to initiate transcription at levels
detectable above
background. In some embodiments, the promoter sequence includes a
transcription initiation site,
as well as protein binding domains responsible for the binding of RNA
polymerase. Eukalyotic
promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.
1002981 In some embodiments, the engineered and/or hypoimmunogenic cells
described are
propagated from a primary T cell or a progeny thereof. As used herein, the
term "propagated
from a primary T cell or a progeny thereof' encompasses the initial primary T
cell that is isolated
from the donor subject and any subsequent progeny thereof. As used herein, the
term "progeny"
encompasses, e.g., a first-generation progeny, i.e., the progeny is directly
derived from, obtained
from, obtainable from or derivable from the initial primary T cell by, e.g.,
traditional propagation
methods. The term "progeny" also encompasses further generations such as
second, third, fourth,
fifth, sixth, seventh, or more generations, i.e., generations of cells which
are derived from,
obtained from, obtainable from or derivable from the former generation by,
e.g., traditional
propagation methods. The term -progeny" also encompasses modified cells that
result from the
modification or alteration of the initial primary T cell or a progeny thereof.
1002991 "Protocadherin-11 "protocadherin-11-Y-, and "PCDH11Y-
and variations
thereof refer to the Y chromosome linked antigen encoded by the PC'D111 1Y
gene.
1003001 As used herein, the terms "regulatory sequences,"
"regulatory elements," and
"control elements" are interchangeable and refer to polynucleotide sequences
that are upstream
(5' non-coding sequences), within, or downstream (3' non-translated sequences)
of a
polynucleotide target to be expressed. Regulatory sequences influence, for
example but are not
limited to, the timing of transcription, amount or level of transcription, RNA
processing or
stability, and/or translation of the related structural nucleotide sequence.
Regulatory sequences
may include activator binding sequences, enhancers, introns, polyadenylation
recognition
sequences, promoters, repressor binding sequences, stem-loop structures,
translational initiation
sequences, translation leader sequences, transcription termination sequences,
translation
termination sequences, primer binding sites, and the like. It is recognized
that since in most cases
44
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
the exact boundaries of regulatory sequences have not been completely defined,
nucleotide
sequences of different lengths may have identical regulatory or promoter
activity.
1003011 "Safe harbor locus" as used herein refers to a gene locus
that allows expression of a
transgene or an exogenous gene in a manner that enables the newly inserted
genetic elements to
function predictably and that also may not cause alterations of the host
genome in a manner that
poses a risk to the host cell. Exemplary "safe harbor" loci include, but are
not limited to, a CCR5
gene, a PPP1R12C (also known as AAVS1) gene, a CLYBL gene, and/or a Rosa gene
(e.g.,
ROSA26).
1003021 "Target locus" as used herein refers to a gene locus that
allows expression of a
transgene or an exogenous gene. Exemplary "target loci" include, but are not
limited to, a
CXCR4 gene, an albumin gene, a SHS231 locus, an F3 gene (also known as CD142),
a MICA
gene, a MICB gene, a LRP1 gene (also known as CD91), a HMGB1 gene, an ABO
gene, a RHD
gene, a FUT I gene, and/or a KDM5D gene (also known as HY). The exogenous
polynucleotide
encoding the exogenous gene can be inserted in the CDS region for B2M, CIITA,
TRAC, TRBC,
CCR5, F3 (i.e., CD142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT I, KDM5D (i.e.,

HY), PDGFRa, OLIG2, and/or GFAP. The exogenous polynucleotide encoding the
exogenous
gene can be inserted in introns 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. The
exogenous
polynucleotide encoding the exogenous gene can be inserted in exons 1 or 2 or
3 for CCR5. The
exogenous polynucleotide encoding the exogenous gene can be inserted in intron
2 for CLYBL.
The exogenous polynucleotide encoding the exogenous gene can be inserted in a
500 bp window
in Ch-4:58,976,613 (i.e., SHS231). The exogenous polynucleotide encoding the
exogenous gene
can be insert in any suitable region of the aforementioned safe harbor or
target loci that allows
for expression of the exogenous, including, for example, an intron, an exon or
a coding sequence
region in a safe harbor or target locus.
1003031 The term "sensitized" used in connection with a patient refers to a
patient that has
antibodies that react to foreign cells. In some embodiments, the present
disclosure contemplates
treatment of sensitized subjects. For example, subjects contemplated for the
present treatment
methods are sensitized to or against one or more alloantigens comprising Y
chromosome linked
antigens. In some embodiments, the patient is sensitized from a previous
pregnancy or a previous
allogeneic transplant (including, for example but not limited to an allogeneic
cell transplant, an
allogeneic blood transfusion, an allogeneic tissue transplant, and an
allogeneic organ transplant).
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
In some embodiments, the patient exhibits memory B cells and/or memory T cells
reactive
against the one or more alloantigens.
1003041 In some embodiments, the present disclosure contemplates treatment of
non-sensitized
subjects. For example, subjects contemplated for the present treatment methods
are not sensitized
to or against one or more alloantigens comprising Y chromosome linked
antigens. In some
embodiments, the patient is not sensitized from a previous pregnancy or a
previous allogeneic
transplant (including, for example but not limited to an allogeneic cell
transplant, an allogeneic
blood transfusion, an allogeneic tissue transplant, and an allogeneic organ
transplant). In some
embodiments, the patient does not exhibit memory B cells and/or memory T cells
reactive
against the one or more alloantigens.
1003051 As used herein, a "target" can refer to a gene, a portion
of a gene, a portion of the
genome, or a protein that is subject to regulatable reduced expression by the
methods described
herein.
1003061 As used herein, "therapeutically effective amount" refers
to an amount sufficient to
provide a therapeutic benefit in the treatment and/or management of a disease,
disorder, or
condition. In some embodiments, a therapeutically effective amount is an
amount sufficient to
ameliorate, palliate, stabilize, reverse, slow, attenuate or delay the
progression of a disease,
disorder, or condition, or of a symptom or side effect of the disease,
disorder, or condition. In
some embodiments, the therapeutically effective amount is also a clinically
effective amount. In
other embodiments, the therapeutically effective amount is not a clinically
effective amount.
1003071 As used herein, the term "treating" and "treatment"
includes administering to a
subject a therapeutically or clinically effective amount of cells described
herein so that the
subject has a reduction in at least one symptom of the disease or an
improvement in the disease,
for example, beneficial or desired therapeutic or clinical results. For
purposes of this technology,
beneficial or desired therapeutic or clinical results include, but are not
limited to, alleviation of
one or more symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state
of disease, delay or slowing of disease progression, amelioration or
palliation of the disease state,
and remission (whether partial or total), whether detectable or undetectable.
Treating can refer to
prolonging survival as compared to expected survival if not receiving
treatment. Thus, one of
skill in the art realizes that a treatment may improve the disease condition,
but may not be a
complete cure for the disease. In some embodiments, one or more symptoms of a
condition,
46
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
disease or disorder are alleviated by at least 5%, at least 10%, at least 20%,
at least 30%, at least
40%, or at least 50% upon treatment of the condition, disease or disorder.
[00308] For purposes of this technology, beneficial or desired
therapeutic or clinical results
of disease treatment include, but are not limited to, alleviation of one or
more symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening) state of
disease, delay or
slowing of disease progression, amelioration or palliation of the disease
state, and remission
(whether partial or total), whether detectable or undetectable.
[00309] A "vector" or "construct" is capable of transferring gene sequences to
target cells.
Typically, "vector construct," "expression vector," and "gene transfer
vector," mean any nucleic
acid construct capable of directing the expression of a gene of interest and
which can transfer
gene sequences to target cells. Thus, the term includes cloning, and
expression vehicles, as well
as integrating vectors. Methods for the introduction of vectors or constructs
into cells are known
to those of skill in the art and include, but are not limited to, lipid-
mediated transfer (i.e.,
liposomes, including neutral and cationic lipids), electroporation, direct
injection, cell fusion,
particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-
mediated transfer and
viral vector-mediated transfer.
[00310] In some embodiments, the cells are engineered to have reduced or
increased expression
of one or more targets relative to an unaltered or unmodified wild-type or
control cell. By -wild-
type" or "wt" or "control" in the context of a cell means any cell found in
nature. Examples of
wild-type or control cells include primary cells and T cells found in nature
However, by way of
example, in the context of an engineered and/or hypoimmunogenic T cell, as
used herein, "wild-
type" or "control" can also mean an engineered and/or hypoimmunogenic T cell
that may contain
nucleic acid changes resulting in reduced expression of one or more MHC class
I molecules
and/or class II molecules and/or T-cell receptors, and/or overexpression of
CD47 proteins, but
did not undergo the gene editing procedures of the present disclosure to
achieve reduced
expression of the one or more Y chromosome genes. For example, as used herein,
"wild-type" or
"control" means an engineered cell that comprises reduced or knocked out
expression of B2M,
CIITA, and/or TRAC. Also as used herein, "wild-type" or "control" means an
engineered cell
that comprises reduced or knocked out expression of B2M, CIITA, TRAC, and/or
TRBC. As
used herein, "wild-type" or "control" also means an engineered cell that may
contain nucleic
acid changes resulting in overexpression of CD47 proteins, but did not undergo
the gene editing
47
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
procedures to result in reduced expression of one or more MTIC class I
molecules and/or class II
molecules and/or T-cell receptors. In the context of an iPSC or a progeny
thereof, -wild-type" or
"control" also means an iPSC or progeny thereof that may contain nucleic acid
changes resulting
in pluripotency but did not undergo the gene editing procedures of the present
disclosure to
achieve reduced expression of one or more MHC class I molecules and/or class
II molecules
and/or T-cell receptors and/or one or more Y chromosome genes, and/or
overexpression of
CD47 proteins. For example, as used herein, "wild-type" or "control" means an
iPSC or progeny
thereof that comprises reduced or knocked out expression of B2M, CIITA, and/or
TRAC. Also
as used herein, "wild-type" or "control" means an iPSC or progeny thereof that
comprises
reduced or knocked out expression of B2M, CIITA, TRAC, and/or TRBC. In the
context of a
primary T cell or a progeny thereof, "wild-type" or "control" also means a
primary T cell or
progeny thereof that may contain nucleic acid changes resulting in reduced
expression of one or
more MHC class I molecules and/or class II molecules and/or T-cell receptors,
but did not
undergo the gene editing procedures of the present disclosure to achieve
reduced expression of
the one or more Y chromosome genes. For example, as used herein, "wild-type"
or "control"
means a primary T cell or progeny thereof that comprises reduced or knocked
out expression of
B2M, CIITA, and/or TRAC, but did not undergo the gene editing procedures to
achieve reduced
expression of the one or more Y chromosome genes. Also as used herein, -wild-
type" or
"control" means a primary T cell or progeny thereof that comprises reduced or
knocked out
expression of B2M, CIITA, FRAC, and/or TRBC, but did not undergo the gene
editing
procedures to achieve reduced expression of the one or more Y chromosome
genes. Also in the
context of a primary T cell or a progeny thereof, "wild-type" or "control"
also means a primary T
cell or progeny thereof that may contain nucleic acid changes resulting in
overexpression of
CD47 proteins, but did not undergo the gene editing procedures to achieve
reduced expression of
the one or more Y chromosome genes. In some embodiments, the wild-type or the
control cell is
a starting material. In some embodiments, the starting material is otherwise
modified or
engineered to have altered expression of one or more genes. In some
embodiments, "wild-type"
or "control" refers to a cell having a Y chromosome.
1003111 As used herein, "Y chromosome linked antigen", "Y chromosome antigen",

"histocompatibility Y chromosome linked antigen", or "male-specific antigen"
and variations
thereof refer to a peptide encoded by a gene on a Y chromosome that is capable
of provoking an
48
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
immune response. In particular, the peptide is capable of provoking an immune
response when
presented in the context of an Mk-IC molecule and/or when antibodies to the
peptide are present.
Y chromosome linked antigens include antigens that are antigenic portions of
or are a whole
protein encoded by a gene on a Y chromosome. Examples of Y chromosome linked
antigens
include, but are not limited to, protocadherin-11 Y-linked (PCDH11Y),
neuroligin-4 Y-linked
(NLGN4Y), H-Y antigen, and the like.
1003121 IL is noted that the claims may be drafted to exclude any optional
element. As such, this
statement is intended to serve as antecedent basis for use of such exclusive
terminology as
"solely," "only," and the like in connection with the recitation of claim
elements, or use of a
"negative" limitation. As will be apparent to those of skill in the art upon
reading this disclosure,
each of the individual embodiments described and illustrated herein has
discrete components and
features readily separated from or combined with the features of any of the
other several
embodiments without departing from the scope or spirit of the present
disclosure. Any recited
method may be carried out in the order of events recited or in any other order
that is logically
possible. Although any methods and materials similar or equivalent to those
described herein
may also be used in the practice or testing of the present disclosure,
representative illustrative
methods and materials are now described.
1003131 Unless defined otherwise, all technical and scientific terms used
herein have the same
meaning as commonly understood by one of ordinary skill in the art to which
this technology
belongs. Where a range of values is provided, it is understood that each
intervening value, to the
tenth of the unit of the lower limit unless the context clearly dictates
otherwise, between the
upper and lower limit of that range and any other stated or intervening value
in that stated range,
is encompassed within the present disclosure. The upper and lower limits of
these smaller ranges
may independently be included in the smaller ranges and are also encompassed
within the
present disclosure, subject to any specifically excluded limit in the stated
range. Where the stated
range includes one or both of the limits, ranges excluding either or both of
those included limits
are also included in the present disclosure. Certain ranges are presented
herein with numerical
values being preceded by the term "about." The term "about" is used herein to
provide literal
support for the exact number that it precedes, as well as a number that is
near to or
approximately the number that the term precedes. In determining whether a
number is near to or
approximately a specifically recited number, the near or approximating
unrecited number may be
49
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
a number, which, in the context presented, provides the substantial equivalent
of the specifically
recited number. The term about is used herein to mean plus or minus ten
percent (10%) of a
value. For example, "about 100" refers to any number between 90 and 110.
1003141 All publications, patents, and patent applications cited in this
specification are
incorporated herein by reference to the same extent as if each individual
publication, patent, or
patent application were specifically and individually indicated to be
incorporated by reference.
Fui theimoie, each cited publication, patent, or patent application is
incorporated herein by
reference to disclose and describe the subject matter in connection with which
the publications
are cited. The citation of any publication is for its disclosure prior to the
filing date and should
not be construed as an admission that the technology described herein is not
entitled to antedate
such publication by virtue of prior technology. Further, the dates of
publication provided might
be different from the actual publication dates, which may need to be
independently confirmed.
1003151 Before the technology is further described, it is to be understood
that this technology is
not limited to particular embodiments described, as such may, of course, vary.
It is also to be
understood that the terminology used herein is for the purpose of describing
particular
embodiments only, and is not intended to be limiting, since the scope of the
present disclosure
will be limited only by the appended claims. It should also be understood that
the headers used
herein are not limiting and are merely intended to orient the reader, but the
subject matter
generally applies to the technology disclosed herein.
III. DETAILED DESCRIPTION
A. Hypoimmunogenic Cells
1003161 In some embodiments, the present disclosure provides engineered (e.g.,
modified and
genetically modified) cells that comprise reduced expression of one or more Y
chromosome
genes and MHC class I and/or MEW class II human leukocyte antigen molecules
relative to an
unaltered or unmodified wild-type or control cell, and a first exogenous
polynucleotide encoding
CD47, wherein the engineered cell is propagated from a primary T cell or a
progeny thereof, an
induced pluripotent stem cell (iPSC) or a progeny thereof. In some
embodiments, the cells are
able to evade activating NK cell mediated and/or antibody-based immune
responses.
1003171 In some embodiments, the cells are induced pluripotent stem cells, any
type of
differentiated cells thereof, primary immune cells and other primary cells of
any tissue. In some
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, the differentiated cells are cardiac cells and subpopulations
thereof, neural cells
and subpopulations thereof, cerebral endothelial cells and subpopulations
thereof, dopaminergic
neurons and subpopulations thereof, glial progenitor cells and subpopulations
thereof,
endothelial cells and subpopulations thereof, thyroid cells and subpopulations
thereof,
hepatocytes and subpopulations thereof, pancreatic islet cells and
subpopulations thereof, or
retinal pigmented epithelium cells and subpopulations thereof. In some
embodiments, the
differentiated cells are T cells and subpopulations thereof, NK cells and
subpopulations thereof,
and endothelial cells and subpopulations thereof. In some embodiments, the
primary immune
cells are T cells and subpopulations thereof and NK cells and subpopulations
thereof. In some
embodiments, the primary tissue cells include primary endothelial cells and
subpopulations
thereof.
1003181 In some embodiments, cells described herein comprise reduced
expression of one or
more Y chromosome genes and MHC class I and/or class II human leukocyte
antigen molecules
relative to an unaltered or unmodified wild-type or control cell. In some
embodiments, cells
described herein comprise reduced expression of Protocadherin-11 Y-linked
and/or Neuroligin-4
Y-linked and MHC class I and/or class II human leukocyte antigen molecules
relative to an
unaltered or unmodified wild-type or control cell. In some embodiments, cells
described herein
comprise reduced expression of Protocadherin-11 Y-linked and MHC class I
and/or class II
human leukocyte antigen molecules relative to an unaltered or unmodified wild-
type or control
cell. In some embodiments, cells described herein comprise reduced expression
of Neuroligin-4
Y-linked and MHC class I and/or class II human leukocyte antigen molecules
relative to an
unaltered or unmodified wild-type or control cell. In some embodiments, cells
described herein
comprise reduced expression of Protocadherin-11 Y-linked and Neuroligin-4 Y-
linked and MEW
class I and/or class II human leukocyte antigen molecules relative to an
unaltered or unmodified
wild-type or control cell. In some embodiments, cells described herein
comprise a first
exogenous polynucleotide encoding CD47. In some embodiments, cells described
herein
comprise a second exogenous polynucleotide encoding a CAR.
1003191 In some embodiments, the present disclosure is directed to pluripotent
stem cells, (e.g.,
pluripotent stem cells and induced pluripotent stem cells (iPSCs)),
differentiated cells derived
from such pluripotent stem cells (such as, but not limited to, T cells, NK
cells, cardiac cells,
neural cells, cerebral endothelial cells, dopaminergic neurons, glial
progenitor cells, endothelial
51
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
cells, thyroid cells, hepatocytes, pancreatic islet cells, and retinal
pigmented epithelium cells),
and primary cells (such as, but not limited to, primary T cells and primary NK
cells). In some
embodiments, the pluripotent stem cells, differentiated cells derived
therefrom such as T cells,
NK cells, cardiac cells, neural cells, cerebral endothelial cells,
dopaminergic neurons, glial
progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic
islet cells, and retinal
pigmented epithelium cells, and primary cells such as primary T cells and
primary NK cells are
engineered for reduced expression or lack of expression of one or more Y
chromosome genes
and MEC class I and/or MHC class II human leukocyte antigen molecules, and in
some
instances, for reduced expression or lack of expression of a T-cell receptor
(TCR) complex. In
some embodiments, the hypoimmune (HIP) T cells and primary T cells overexpress
CD47 and a
chimeric antigen receptor (CAR) in addition to reduced expression or lack of
expression of one
or more Y chromosome genes and MHC class I and/or MHC class II human leukocyte
antigen
molecules, and have reduced expression or lack expression of a T-cell receptor
(TCR) complex.
In some embodiments, the CAR comprises an antigen binding domain that binds to
any one
selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138,
and BCMA.
In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the
CAR is a
CD20-specific CAR. In some embodiments, the CAR is a CD22-specific CAR. In
some
instances, the CAR is a CD38-specific CAR. In some embodiments, the CAR is a
CD123-
specific CAR. In some embodiments, the CAR is a CD138-specific CAR. In some
instances,
the CAR is a BCMA-specific CAR. In some embodiments, the CAR is a bispecific
CAR. In
some embodiments, the bispecific CAR is a CD19/CD20-bispecific CAR. In some
embodiments, the bispecific CAR is a CD19/CD22-bispecific CAR. In some
embodiments, the
bispecific CAR is a BCMA/CD38-bispecific CAR. In some embodiments, the cells
described
express a CD19-specific CAR and a different CAR, such as, but not limited to a
CD20-specific
CAR, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-
specific
CAR, and a BCMA-specific CAR. In some embodiments, the cells described express
a CD20-
specific CAR and a different CAR, such as, but not limited to a a CD22-
specific CAR, a CD38-
specific CAR, a CD123-specific CAR, a CD138-specific CAR, a CD19-specific CAR,
and a
BCMA-specific CAR. In some embodiments, the cells described express a CD22-
specific CAR
and a different CAR, such as, but not limited to a CD19-specific CAR, a CD20-
specific CAR, a
CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-
specific
52
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CAR. In some embodiments, the cells described express a CD38-specific CAR and
a different
CAR, such as, but not limited to a CD20-specific CAR, a CD22-specific CAR, a
CD18-specific
CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In
some
embodiments, the cells described express a CD123-specific CAR and a different
CAR, such as,
but not limited to a CD20-specific CAR, a CD22-specific CAR, a CD38-specific
CAR, a CD19-
specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some
embodiments, the
cells described express a CD138-specific CAR and a different CAR, such as, but
not limited to a
CD20-specific CAR, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific
CAR, a
CD19-specific CAR, and a BCMA-specific CAR. In some embodiments, the cells
described
express a BCMA-specific CAR and a different CAR, such as, but not limited to a
CD20-specific
CAR, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-
specific
CAR, and a CD19-specific CAR.
1003201 In some embodiments, hypoimmune cells derived from iPSCs, such as, but
not limited
to, T cells, NK cells, cardiac cells, neural cells, cerebral endothelial
cells, dopaminergic neurons,
glial progenitor cells, endothelial cells, thyroid cells, hepatocytes,
pancreatic islet cells, and
retinal pigmented epithelium cells, overexpress CD47, and include a gcnomic
modification or
knock out or knock down of the PCDH11Y gene. In some embodiments, hypoimmune
cells
derived from iPSCs, such as, but not limited to, T cells, NK cells, cardiac
cells, neural cells,
cerebral endothelial cells, dopaminergic neurons, glial progenitor cells,
endothelial cells, thyroid
cells, hepatocytes, pancreatic islet cells, and retinal pigmented epithelium
cells, overexpress
CD47, and include a genomic modification or knock out or knock down of the
NLGN4Y gene.
In some embodiments, hypoimmune cells derived from iPSCs, such as, but not
limited to, T
cells, NK cells, cardiac cells, neural cells, cerebral endothelial cells,
dopaminergic neurons, glial
progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic
islet cells, and retinal
pigmented epithelium cells, overexpress CD47, and include a genomic
modification or knock out
or knock down of the B2M gene. In some embodiments, hypoimmune cells derived
from iPSCs,
such as, but not limited to, T cells, NK cells, cardiac cells, neural cells,
cerebral endothelial cells,
dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid
cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells, overexpress
CD47 and include a
genomic modification or knock out or knock down of the CIITA gene. In some
embodiments,
the cells are PCDHIIY, NLGN417-/-, B2M, CIITA, CD47tg cells. In some
embodiments, the
53
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
cells are PCDH11Y-/-, NLGN4Y-/-, B211,1, CD47tg cells. In some embodiments,
the cells are
PCDH1117mcieUendel, NLGN4Yindel/Indel, B2m1ndelandel cHTAindel/endel, CD47tg
cells. In some
embodiments, the cells are /'CDHllYindelandel, NLGN4rndePindel,
B2m1nde1/inde1, CD47tg cells. In
some embodiments, the cells are PCDHllYk
Hock down , ATGN-4yknock down B21vIkn0ck down chTTAknock
down , CD47tg cells. In some embodiments, the cells are PCDH 1 lYk
nock down , NLGN4yknock down ,
B2mknock down CD47tg cells. In some embodiments, hypoimmune cells derived from
iPSCs are
produced by differentiating induced pluripotent stem cells such as
hypoimmunogenic induced
pluripotent stem cells. In some embodiments, the cells are modified or
engineered as compared
to a wild-type or control cell, including an unaltered or unmodified wild-type
cell or control cell.
In some embodiments, the wild-type cell or the control cell is a starting
material. In some
embodiments, the starting material is otherwise modified or engineered to have
altered
expression of one or more genes to generate the engineered cell.
1003211 In some embodiments, hypoimmune cells derived from ESCs, such as, but
not limited
to, T cells, NK cells, cardiac cells, neural cells, cerebral endothelial
cells, dopaminergic neurons,
glial progenitor cells, endothelial cells, thyroid cells, hepatocytes,
pancreatic islet cells, and
retinal pigmented epithelium cells, overexpress CD47, and include a gcnomic
modification or
knock out or knock down of the PCDH11Y gene. In some embodiments, hypoimmune
cells
derived from ESCs, such as, but not limited to, T cells, NK cells, cardiac
cells, neural cells,
cerebral endothelial cells, dopaminergic neurons, glial progenitor cells,
endothelial cells, thyroid
cells, hepatocytes, pancreatic islet cells, and retinal pigmented epithelium
cells, overexpress
CD47, and include a genomic modification or knock out or knock down of the
NLGN4Y gene.
In some embodiments, hypoimmune cells derived from ESCs, such as, but not
limited to, T cells,
NK cells, cardiac cells, neural cells, cerebral endothelial cells,
dopaminergic neurons, glial
progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic
islet cells, and retinal
pigmented epithelium cells, overexpress CD47, and include a genomic
modification or knock out
or knock down of the B2M gene. In some embodiments, hypoimmune cells derived
from ESCs,
such as, but not limited to, T cells, NK cells, cardiac cells, neural cells,
cerebral endothelial cells,
dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid
cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells, overexpress
CD47 and include a
genomic modification or knock out or knock down of the CIITA gene. In some
embodiments,
the cells are PCDHIIY, NLGN4Y-/-, CD47tg cells. In some
embodiments, the
54
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
cells are PCDH11Y-/-, NLGN4Y, B211,1, CD47tg cells. In some embodiments, the
cells are
PCDH11YindeUindel, NLGN4Yindel/indel B2m1ndek1ndel cHTAindel/indel, CD47tg
cells. In some
embodiments, the cells are /'CDHllYindelandel, NLGN4Pncleuindel,
B2m1nde1/inde1 CD47tg cells. In
some embodiments, the cells are PCDHllYk
Hock down , ATGN-4yknock down B21vIkn0ck down chTTAknock
down , CD47tg cells. In some embodiments, the cells are PCDH 1 lYk
nock down , NLGN4yknock down ,
B2mknock down CD47tg cells. In some embodiments, hypoimmune cells derived from
iPSCs are
produced by differentiating pluripotent stem cells such as hypoimmunogenic
embryonic stem
cells. In some embodiments, the cells are modified or engineered as compared
to a wild-type or
control cell, including an unaltered or unmodified wild-type cell or control
cell. In some
embodiments, the wild-type cell or the control cell is a starting material. In
some embodiments,
the starting material is otherwise modified or engineered to have altered
expression of one or
more genes to generate the engineered cell.
1003221 In some embodiments, hypoimmune (HIP) T cells derived from iPSCs and
primary T
cells overexpress CD47 and a chimeric antigen receptor (CAR), and include a
genomic
modification or knock out or knock down of the PCDHIIY gene. In some
embodiments,
hypoimmune (HIP) T cells derived from iPSCs and primary T cells overexpress
CD47 and a
chimeric antigen receptor (CAR), and include a genomic modification or knock
out or knock
down of the NLGN4Y gene. In some embodiments, hypoimmune (HIP) T cells derived
from
iPSCs and primary T cells overexpress CD47 and a chimeric antigen receptor
(CAR), and
include a genomic modification or knock out or knock down of the B2M gene. In
some
embodiments, hypoimmune (HIP) T cells derived from iPSCs and primary T cells
overexpress
CD47 and include a genomic modification or knock out or knock down of the
CIITA gene. In
some embodiments, hypoimmune (HIP) T cells derived from iPSCs and primary T
cells
overexpress CD47 and a CAR, and include a genomic modification or knock out or
knock down
of the TRAC gene. In some embodiments, hypoimmune (HIP) T cells derived from
iPSCs and
primary T cells overexpress CD47 and a CAR, and include a genomic modification
or knock out
or knock down of the TRB gene. In some embodiments, hypoimmune (HIP) T cells
derived
from iPSCs and primary T cells overexpress CD47 and a CAR, and include one or
more genomic
modifications or knock outs or knock downs selected from the group consisting
of the
PCDH11Y, NLGN4Y, B2M, CIITA, TRAC, and TRB genes. In some embodiments,
hypoimmune (HIP) T cells derived from iPSCs and primary T cells overexpress
CD47 and a
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CAR, and include genomic modifications or knock outs or knock downs of the
PCDHilY,
NLGN4Y, B2M, CIITA, TRAC, and IRE genes. In some embodiments, the cells are
B211/1-,
CIITA", TRAC", CD47tg cells that also express CARs. In some embodiments, the
cells are
132A/1, TRAC', CD47tg cells that also express CARs. In some embodiments,
hypoimmune
(HIP) T cells are produced by differentiating induced pluripotent stem cells
such as
hypoimmunogenic induced pluripotent stem cells. In some embodiments, the cells
are modified
or engineered as compared to a wild-type or control cell, including an
unaltered or unmodified
wild-type cell or control cell. In some embodiments, the wild-type cell or the
control cell is a
starting material. In some embodiments, the starting material is otherwise
modified or
engineered to have altered expression of one or more genes to generate the
engineered cell.
1003231 In some embodiments, the hypoimmune (HIP) T cells derived from iPSCs
and primary
T cells are PCDH TY", NLGN4Y", B2M", CHTA", TRB", CD47tg cells that also
express
CARs. In some embodiments, the hypoimmune (HIP) T cells derived from iPSCs and
primary T
cells are PCDH 1 IY", NIGN4Y", B21V1, TR13", CD47tg cells that also express
CARs. In some
embodiments, the cells are PCDH11Y, NLGN4Y",B2M", CHTA", TRAC", TRB", CD47tg
cells that also express CARs. In some embodiments, the cells are PCDH IY",
NLGN4Y",B2111-
/ , TRAC", TRU", CD47tg cells that also express CARs. In certain embodiments,
the cells are
PCDH11Yindel/inciel , NLGN4 Yindeldndel B2minde171nde1 CHTAindeldndel
TRACEndel/indel CD47tg cells that
also express CARs. In certain embodiments, the cells are PCDH 1 llYndelAndel,
NIG7j4Yincie1/indel ,
B2mndel/indel TRACY'', CD47tg cells that also express CARs. In certain
embodiments, the
cells are PCDH11rndelAndel , NLGN4Y`ndel/indel BAIndeldndel
CHTAIndellAndel TRIrdel/Andel, CD47tg
cells that also express CARs. In certain embodiments, the cells are PCDH
lYindelfindel
NLGN4Yindel/indel B2mindel/indel TRBindel/indel CD47tg- cells that also
express CARs. In certain
embodiments, the cells are PCDH1 1Yi ndellindel , NIGN4Yind'uindel,
B211/Pnde1/indel cHTAindel/indel
TRAcindel/indel, TRBindel/indel, CD47tg cells that also express CARs. In
certain embodiments, the
cells are PCDH I lYindel/indel NLGN4rndel/indel, B21/linde1/indel
TRAcindel/indel, TRBindeldndel CD47tg
cells that also express CARs. In some embodiments, the cells are PCDH lYk"ack
d'w",
NLGN4Yk
nock down, R21knock down
CIITAknock down TRAcknock down, CD47tg cells that also express
CARs. In certain embodiments, the cells are PCDH lYk
""k claw", NLGN4Y1c
nock down, B2mknock, down
cHTAknock down , TRBknock down, CD47tg cells that also express CARs. In
certain embodiments, the
cells are PCDHI lYknock down NLGN4Yknock down, R2 1icnock down CHTAknocic down
TRAcknock down ,
56
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
TRBknock down , CD47tg cells that also express CARs. In some embodiments, the
cells are
PCDHllYkn'k dm", NLGN4Ylmock down, B2A40n0ck down , TRAcknock down , CD47tg
cells that also
express CARs. In certain embodiments, the cells are /,(7DH1 iyknock down, NLGN
4yknock down,
B2mknock down , TRBknock down , CD47tg cells that also express CARs. In
certain embodiments, the
cells are PCDHllYkiwcic down , NLGN4Yk
nock down, B2A4knock down TRAcknock down TRBknock down ,
CD47tg cells that also express CARs. In some embodiments, the cells are
modified or
engineered as compared to a wild-type or control cell, including an unaltered
or unmodified
wild-type cell or control cell. In some embodiments, the wild-type cell or the
control cell is a
starting material. In some embodiments, the starting material is otherwise
modified or
engineered to have altered expression of one or more genes to generate the
engineered cell.
1003241 In some embodiments, the engineered or modified cells described are
pluripotent stem
cells, induced pluripotent stem cells, NK cells differentiated from such
pluripotent stem cells and
induced pluripotent stem cells, T cells differentiated from such pluripotent
stem cells and
induced pluripotent stem cells, or primary T cells. Non-limiting examples of
primary T cells
include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T
(Treg) cells, non-
regulatory T cells, Thl cells, Th2 cells, Th9 cells, Th17 cells, T-follicular
helper (Tfh) cells,
cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm)
cells, effector
memory T (Tem) cells, effector memory T cells express CD45RA (TEMRA cells),
tissue-
resident memory (Trm) cells, virtual memory T cells, innate memory T cells,
memory stem cell
(Tsc), y8 T cells, and any other subtype of T cells. In some embodiments, the
primary T cells are
selected from a group that includes cytotoxic T-cells, helper T-cells, memory
T-cells, regulatory
T-cells, tumor infiltrating lymphocytes, and combinations thereof. Non-
limiting examples of NK
cells and primary NK cells include immature NK cells and mature NK cells. In
some
embodiments, the cells are modified or engineered as compared to a wild-type
or control cell,
including an unaltered or unmodified wild-type cell or control cell. In some
embodiments, the
wild-type cell or the control cell is a starting material. In some
embodiments, the starting
material is otherwise modified or engineered to have altered expression of one
or more genes to
generate the engineered cell.
1003251 In some embodiments, the primary T cells are from a pool of primary T
cells from one
or more donor subjects that are different than the recipient subject (e.g.,
the patient administered
the cells). The primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 20, 50, 100 or
57
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
more donor subjects and pooled together. The primary T cells can be obtained
from 1 or more, 2
or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9
or more, 10, or more
20 or more, 50 or more, or 100 or more donor subjects and pooled together. In
some
embodiments, the primary T cells are harvested from one or a plurality of
individuals, and in
some instances, the primary T cells or the pool of primary T cells are
cultured in vitro. In some
embodiments, the primary T cells or the pool of primary T cells are engineered
to exogenously
express CD47 and cultured in vitro.
1003261 In certain embodiments, the primary T cells or the pool of primary T
cells are
engineered to express a chimeric antigen receptor (CAR). The CAR can be any
known to those
skilled in the art. Useful CARs include those that bind an antigen selected
from a group that
includes CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the
CAR is
the same or equivalent to those used in FDA-approved CAR-T cell therapies such
as, but not
limited to, those used in tisagenlecleucel and axicabtagene ciloleucel, or
others under
investigation in clinical trials.
1003271 In some embodiments, the primary T cells or the pool of primary T
cells are engineered
to exhibit reduced expression of an endogenous T cell receptor compared to
unmodified primary
T cells. In certain embodiments, the primary T cells or the pool of primary T
cells are engineered
to exhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1, as
compared to
unmodified primary T cells. Methods of genetically modifying a cell including
a T cell are
described in detail, for example, in W02020/018620 and W02016/183041, the
disclosures of
which are herein incorporated by reference in their entireties, including the
tables, appendices,
sequence listing and figures.
1003281 In some embodiments, the CAR-T cells comprise a CAR selected from a
group
including: (a) a first generation CAR comprising an antigen binding domain, a
transmembrane
domain, and a signaling domain; (b) a second generation CAR comprising an
antigen binding
domain, a transmembrane domain, and at least two signaling domains; (c) a
third generation
CAR comprising an antigen binding domain, a transmembrane domain, and at least
three
signaling domains; and (d) a fourth generation CAR comprising an antigen
binding domain, a
transmembrane domain, three or four signaling domains, and a domain which upon
successful
signaling of the CAR induces expression of a cytokine gene.
58
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00329] In some embodiments, the CAR-T cells comprise a CAR comprising an
antigen binding
domain, a transmembrane, and one or more signaling domains. In some
embodiments, the CAR
also comprises a linker. In some embodiments, the CAR comprises a CD19 antigen
binding
domain. In some embodiments, the CAR comprises a CD20 antigen binding domain.
In some
embodiments, the CAR comprises a CD22 binding domain. In some embodiments, the
CAR
comprises a BCMA binding domain. In some embodiments, the CAR comprises a CD28
or a
CD8u, transmembrane domain. In some embodiments, the CAR comprises a CD8ot
signal
peptide. In some embodiments, the CAR comprises a Whitlow linker
GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the antigen binding
domain of the CAR is selected from a group including, but not limited to, (a)
an antigen binding
domain targets an antigen characteristic of a neoplastic cell; (b) an antigen
binding domain that
targets an antigen characteristic of a T cell; (c) an antigen binding domain
targets an antigen
characteristic of an autoimmune diseases/disorders and/or inflammatory
diseases/disorders; (d)
an antigen binding domain that targets an antigen characteristic of senescent
cells; (e) an antigen
binding domain that targets an antigen characteristic of an infectious
disease; and (f) an antigen
binding domain that binds to a cell surface antigen of a cell.
[00330] In some embodiments, the CAR further comprises one or more
linkers. The format
of an scFv is generally two variable domains linked by a flexible peptide
sequence, or a "linker,"
either in the orientation VI-linker-VL or VL-linker-VH. Any suitable linker
known to those in
the art in view of the specification can be used in the CARs. Examples of
suitable linkers
include, but are not limited to, a GS based linker sequence, and a Whitlow
linker
GSTSGSGKPGSGEGSTKG (SEQ ID NO:15). In some embodiments, the linker is a GS or
a
gly-ser linker. Exemplary gly-ser polypeptide linkers comprise the amino acid
sequence
Ser(Gly4Ser)n, as well as (Gly4Ser)n and/or (Gly4Ser3)n. In some embodiments,
n=1. In some
embodiments, n=2. In some embodiments, n=3, i.e., Ser(G1y4Ser)3. In some
embodiments, n=4,
i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In
some
embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some
embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the
amino acid
sequence Ser(Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In
some
embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In
some
embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises
(Gly4Ser)n. In
59
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In
some
embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another
exemplary
gly-ser polypeptide linker comprises (Gly3Ser)n. In some embodiments, n=1. In
some
embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In
another
embodiment, n=5. In yet another embodiment, n=6. Another exemplary gly-ser
polypeptide
linker comprises (Gly4Ser3)n. In some embodiments, n=1. In some embodiments,
n=2. In some
embodiments, n-3. In some embodiments, n-4. In some embodiments, n-5. In some
embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises
(Gly3Ser)n. In some
embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some
embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6.
1003311 In some embodiments, the antigen binding domain is selected from a
group that
includes an antibody, an antigen-binding portion or fragment thereof, an scFv,
and a Fab. In
some embodiments, the antigen binding domain binds to CD19, CD20, CD22, CD38,
CD123,
CD138, or BCMA. In some embodiments, the antigen binding domain is an anti-
CD19 scFv
such as but not limited to FMC63. In some embodiments, the antigen binding
domain is an anti-
CD20 scFv. In some embodiments, the antigen binding domain is an anti-CD22
scFv. In some
embodiments, the antigen binding domain is an anti-BCMA scFv.
1003321 In some embodiments, the transmembrane domain comprises one selected
from a
group that includes a transmembrane region of TCRct, TCR13, TCRC, CD3E, CD37,
CD36, CD3C,
CD4, CD5, CD8ct, CD8I3, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40,
CD4OL/CD154, CD45, CD64, CD80, CD86, 0X40/CD134, 4-1BB/CD137, CD154, FcERIy,
VEGFR2, FAS, FGFR2B, and functional variant thereof.
1003331 In some embodiments, the signaling domain(s) of the CAR comprises a
costimulatory
domain(s). For instance, a signaling domain can contain a costimulatory
domain. Or, a
signaling domain can contain one or more costimulatory domains. In certain
embodiments, the
signaling domain comprises a costimulatory domain. In other embodiments, the
signaling
domains comprise costimulatory domains. In some cases, when the CAR comprises
two or more
costimulatory domains, two costimulatory domains are not the same. In some
embodiments, the
costimulatory domains comprise two costimulatory domains that are not the
same. In some
embodiments, the costimulatory domain enhances cytokine production, CAR-T cell
proliferation,
and/or CAR-T cell persistence during T cell activation. In some embodiments,
the costimulatory
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
domains enhance cytokine production, CAR-T cell proliferation, and/or CAR-T
cell persistence
during T cell activation.
[00334] As described herein, a fourth generation CAR can contain an antigen
binding domain, a
transmembrane domain, three or four signaling domains, and a domain which upon
successful
signaling of the CAR induces expression of a cytokine gene. In some instances,
the cytokine
gene is an endogenous or exogenous cytokine gene of the engineered and/or
hypoimmunogenic
cells. In some cases, the cytokine gene encodes a pro-inflammatory cytokine.
In some
embodiments, the pro-inflammatory cytokine is selected from a group that
includes IL-1, IL-2,
IL-9, IL-12, IL-18, TNF, IFN-gamma, and a functional fragment thereof. In some
embodiments,
the domain which upon successful signaling of the CAR induces expression of
the cytokine gene
comprises a transcription factor or functional domain or fragment thereof
[00335] In some embodiments, the CAR comprises a CD3 zeta (CD3) domain or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof. In some
embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor
tyrosine-based
activation motif (ITAM), or functional variant thereof; and (ii) a CD28
domain, or a 4-BB
domain, or functional variant thereof. In other embodiments, the CAR comprises
(i) a CD3 zeta
domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or
functional variant
thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB
domain, or a CD134
domain, or functional variant thereof. In certain embodiments, the CAR
comprises (i) a CD3
zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or
functional
variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-
1BB domain, or a
CD134 domain, or functional variant thereof; and (iv) a cytokine or
costimulatory ligand
transgene. In some embodiments, the CAR comprises a (i) an anti-CD19 scFv,
(ii) a CD8a
hinge and transmembrane domain or functional variant thereof; (iii) a 4-1BB
costimulatory
domain or functional variant thereof; and (iv) a CD3 signaling domain or
functional variant
thereof.
[00336] Methods for introducing a CAR construct or producing a CAR-T cells are
well known
to those skilled in the art. Detailed descriptions are found, for example, in
Vormittag et al., Curr
Opin Biotechnol, 2018, 53, 162-181; and Eyquem et al., Nature, 2017, 543, 113-
117.
[00337] In some embodiments, the cells derived from primary T cells comprise
reduced
expression of an endogenous T cell receptor, for example by disruption of an
endogenous T cell
61
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
receptor gene (e.g., T cell receptor alpha constant region (TRAC) or T cell
receptor beta constant
region (TRB)). In some embodiments, an exogenous nucleic acid encoding a
polypeptide as
disclosed herein (e.g., a chimeric antigen receptor, CD47, or another
tolerogenic factor disclosed
herein) is inserted at the disrupted T cell receptor gene. In some
embodiments, an exogenous
nucleic acid encoding a polypeptide is inserted at a TRAC or a TRB gene locus
1003381 In some embodiments, the cells derived from primary T cells comprise
reduced
expression of cy totoxic T-lymphocyte-associated protein 4 (CTLA4) and/or
programmed cell
death (PD1). Methods of reducing or eliminating expression of CTLA4, PD1 and
both CTLA4
and PD1 can include any recognized by those skilled in the art, such as but
not limited to, genetic
modification technologies that utilize rare-cutting endonucleases and RNA
silencing or RNA
interference technologies. Non-limiting examples of a rare-cutting
endonuclease include any
Cas protein, TALEN, zinc finger nuclease, meganuclease, and/or homing
endonuclease. In some
embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed
herein (e.g., a
chimeric antigen receptor, CD47, or another tolerogenic factor disclosed
herein) is inserted at a
CTLA4 and/or PD1 gene locus.
1003391 In some embodiments, a CD47 transgene is inserted into a pre-selected
locus of the
cell. In some embodiments, a transgene encoding a CAR is inserted into a pre-
selected locus of
the cell. In certain embodiments, a CD47 transgene and a transgene encoding a
CAR are
inserted into a pre-selected locus of the cell. The pre-selected locus can be
a safe harbor or a
target locus. Non-limiting examples of a safe harbor or target locus include,
but are not limited
to, a CCR5 gene locus, a PPP1R12C (also known as AAVS I) gene locus, a CLYBL
gene locus,
and a Rosa gene locus (e.g., ROSA26 gene locus). Non-limiting examples of a
target locus
include, but are not limited to, a CXCR4 gene locus, an albumin gene locus, a
SHS231 gene
locus, an F3 gene locus (also known as CD142), a MICA gene locus, a MICB gene
locus, a
LRP1 gene locus (also known as a CD91 gene locus), a 1-1MGB1 gene locus, an
ABO gene locus,
a RHD gene locus, a FUT1 locus, and a KDM5D gene locus. The CD47 transgene can
be
inserted in Introns 1 or 2 for PPP1R12C (i.e., AAVS1) or CCR5. The CD47
transgene can be
inserted in Exons 1 or 2 or 3 for CCR5. The CD47 transgene can be inserted in
intron 2 for
CLYBL. The CD47 transgene can be inserted in a 500 bp window in Ch-
4:58,976,613 (i.e.,
SHS231). The CD47 transgene can be insert in any suitable region of the
aforementioned safe
harbor or target loci that allows for expression of the exogenous, including,
for example, an
62
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
intron, an exon or a coding sequence region in a safe harbor or target locus.
In some
embodiments, the pre-selected locus is selected from the group consisting of
the B2M locus, the
(7111A locus, the TRAC locus, and the 'FRB locus. In some embodiments, the pre-
selected locus is
the B2M- locus. In some embodiments, the pre-selected locus is the CIITA
locus. In some
embodiments, the pre-selected locus is the TRAC locus. In some embodiments,
the pre-selected
locus is the TRB locus.
1003401 In some embodiments, a CD47 transgene and a transgene encoding a CAR
are inserted
into the same locus. In some embodiments, a CD47 transgene and a transgene
encoding a CAR
are inserted into different loci. In many instances, a CD47 transgene is
inserted into a safe
harbor or target locus. In many instances, a transgene encoding a CAR is
inserted into a safe
harbor or target locus. In some instances, a CD47 transgene is inserted into a
B2M locus. In
some instances, a transgene encoding a CAR is inserted into a B2M locus. In
certain instances, a
CD47 transgene is inserted into a CIITA locus. In certain instances, a
transgene encoding a CAR
is inserted into a CIITA locus. In particular instances, a CD47 transgene is
inserted into a TRAC
locus. In particular instances, a transgene encoding a CAR is inserted into a
TRAC locus. In
many other instances, a CD47 transgene is inserted into a TRB locus. In many
other instances, a
transgene encoding a CAR is inserted into a TRB locus. In some embodiments, a
CD47
transgene and a transgene encoding a CAR are inserted into a safe harbor or
target locus (e.g., a
CCR5 gene locus, a CXCR4 gene locus, a PPP1R12C gene locus, an albumin gene
locus, a
SHS231 gene locus, a CLYBL gene locus, a Rosa gene locus, an F3 (CD142) gene
locus, a
MICA gene locus, a MICB gene locus, a LRP1 (CD91) gene locus, a HMGB1 gene
locus, an
ABO gene locus, a RHD gene locus, a FUT1 locus, and a KDM5D gene locus.
1003411 In certain embodiments, a CD47 transgene and a transgene encoding a
CAR are
inserted into a safe harbor or target locus. In certain embodiments, a CD47
transgene and a
transgene encoding a CAR are controlled by a single promoter and are inserted
into a safe harbor
or target locus. In certain embodiments, a CD47 transgene and a transgene
encoding a CAR are
controlled by their own promoters and are inserted into a safe harbor or
target locus. In certain
embodiments, a CD47 transgene and a transgene encoding a CAR are inserted into
a TRAC
locus. In certain embodiments, a CD47 transgene and a transgene encoding a CAR
are
controlled by a single promoter and are inserted into a TRAC locus. In certain
embodiments, a
CD47 transgene and a transgene encoding a CAR are controlled by their own
promoters and are
63
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
inserted into a TRAC locus. In some embodiments, a CD47 transgene and a
transgene encoding
a CAR are inserted into a TRB locus. In some embodiments, a CD47 transgene and
a transgene
encoding a CAR are controlled by a single promoter and are inserted into a TRB
locus. In some
embodiments, a CD47 transgene and a transgene encoding a CAR are controlled by
their own
promoters and are inserted into a TRB locus. In other embodiments, a CD47
transgene and a
transgene encoding a CAR are inserted into a B2M locus. In other embodiments,
a CD47
transgene and a transgene encoding a CAR are controlled by a single promoter
and are inserted
into a B2M locus. In other embodiments, a CD47 transgene and a transgene
encoding a CAR are
controlled by their own promoters and are inserted into a B2M locus. In
various embodiments, a
CD47 transgene and a transgene encoding a CAR are inserted into a CIITA locus.
In various
embodiments, a CD47 transgene and a transgene encoding a CAR are controlled by
a single
promoter and are inserted into a CIITA locus. In various embodiments, a CD47
transgene and a
transgene encoding a CAR are controlled by their own promoters and are
inserted into a CIITA
locus.
1003421 In some instances, the promoter controlling expression of any
transgene described is a
constitutive promoter. In other instances, the promoter for any transgene
described is an
inducible promoter. In some embodiments, the promoter is an EFla promoter. In
some
embodiments, the promoter is CAG promoter. In some embodiments, a CD47
transgene and a
transgene encoding a CAR are both controlled by a constitutive promoter. In
some
embodiments, a CD47 transgene and a transgene encoding a CAR are both
controlled by an
inducible promoter. In some embodiments, a CD47 transgene is controlled by a
constitutive
promoter and a transgene encoding a CAR is controlled by an inducible
promoter. In some
embodiments, a CD47 transgene is controlled by an inducible promoter and a
transgene encoding
a CAR is controlled by a constitutive promoter. In various embodiments, a CD47
transgene is
controlled by an EFla promoter and a transgene encoding a CAR is controlled by
an EFla
promoter. In some embodiments, a CD47 transgene is controlled by a CAG
promoter and a
transgene encoding a CAR is controlled by a CAG promoter. In some embodiments,
a CD47
transgene is controlled by a CAG promoter and a transgene encoding a CAR is
controlled by an
EFla promoter. In some embodiments, a CD47 transgene is controlled by an EFla
promoter
and a transgene encoding a CAR is controlled by a CAG promoter. In some
embodiments,
expression of both a CD47 transgene and a transgene encoding a CAR is
controlled by a single
64
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
EFla promoter. In some embodiments, expression of both a CD47 transgene and a
transgene
encoding a CAR is controlled by a single CAG promoter.
[00343] In another embodiment, the present disclosure disclosed herein is
directed to
pluripotent stem cells, (e.g., pluripotent stem cells and induced pluripotent
stem cells (iPSCs)),
differentiated cells derived from such pluripotent stem cells (e.g.,
hypoimmune (HIP) T cells,
NK cells, cardiac cells, neural cells, cerebral endothelial cells,
dopaminergic neurons, glial
progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic
islet cells, and retinal
pigmented epithelium cells), and primary T cells that overexpress CD47 (such
as exogenously
express CD47 proteins), have reduced expression or lack expression of MEW
class I and/or
MEW class II human leukocyte antigen molecules, and have reduced expression or
lack
expression of a T-cell receptor (TCR) complex. In some embodiments, the
hypoimmune (HIP) T
cells and primary T cells overexpress CD47 (such as exogenously express CD47
proteins), have
reduced expression or lack expression of one or more MI-IC class I and/or MEW
class II human
leukocyte antigen molecules, and have reduced expression or lack expression of
a T-cell receptor
(TCR) complex.
[00344] In some embodiments, pluripotent stem cells, (e.g., pluripotent stem
cells and induced
pluripotent stem cells (iPSCs)), differentiated cells derived from such
pluripotent stem cells (e.g.,
hypoimmune (HIP) T cells, NK cells, cardiac cells, neural cells, cerebral
endothelial cells,
dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid
cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells), and primary T
cells overexpress
CD47 and include a genomic modification of the B2M gene. In some embodiments,
pluripotent
stem cells, differentiated cell derived from such pluripotent stem cells and
primary T cells
overexpress CD47 and include a genomic modification of the CIITA gene. In some

embodiments, the pluripotent stem cells, differentiated cells derived from
such pluripotent stem
cells, such as, but not limited to, T cells, NK cells, cardiac cells, neural
cells, cerebral endothelial
cells, dopaminergic neurons, glial progenitor cells, endothelial cells,
thyroid cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells, are PCDH1117-/-
, NLGN417-/-,
CD47tg cells. In some embodiments, the cells are PCDH11Y", NLGN4Y",
CD47tg cells. In some embodiments, the cells are PCDH//r"dei, NLGN-
117`"del/1"del,
B2mitidel/aidel, CIITAmde"del, CD47tg cells. In some embodiments, the cells
are PCDH Irtidel/eridel,
NLGN4yricielfindel, B2mndemando, CD47tg cells. In some embodiments, the cells
are PCDH//Ykrmck
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
down, NLGN4Y'k down, B2mknock down
CIITAknock down, CD47tg cells. In some embodiments, the
cells are PCDHllYkn'k d'wn, NLGN4Yk
nock down, B2mkn0ck down , CD47tg cells. In some
embodiments, pluripotent stem cells, T cells differentiated from such
pluripotent stem cells and
primary T cells overexpress CD47 and include a genomic modification of the
PCDH11Y gene.
In some embodiments, pluripotent stem cells, T cells differentiated from such
pluripotent stem
cells and primary T cells overexpress CD47 and include a genomic modification
of the NLGN4Y
gene. In sonic embodiments, pluripotent stem cells, T cells differentiated
from such pluripotent
stem cells and primary T cells overexpress CD47 and include a genomic
modification of the
TRAC gene. In some embodiments, pluripotent stem cells, T cells differentiated
from such
pluripotent stem cells and primary T cells overexpress CD47 and include a
genomic modification
of the TRB gene. In some embodiments, pluripotent stem cells, T cells
differentiated from such
pluripotent stem cells and primary T cells overexpress CD47 and include one or
more genomic
modifications selected from the group consisting of the PCDH11Y, NLGN4Y, B2M,
CIITA,
TRAC and TRB genes. In some embodiments, pluripotent stem cells, T cells
differentiated from
such pluripotent stem cells and primary T cells overexpress CD47 and include
genomic
modifications of the PCDH11Y, NLGN4Y, B2M, CIITA and TRAC genes. In some
embodiments, pluripotent stem cells, T cells differentiated from such
pluripotent stem cells and
primary T cells overexpress CD47 and include genomic modifications of the
PCDH11Y,
NLGN4Y, B2M, CIITA and TRB genes. In some embodiments, pluripotent stem cells,
T cells
differentiated from such pluripotent stem cells and primary T cells
overexpress CD47 and
include genomic modifications of the PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB

genes. In certain embodiments, the pluripotent stem cells, differentiated cell
derived from such
pluripotent stem cells and primary T cells are PCDH11Y-/-, NLGN4Y, B211/1/ ,
CIITA', TRAC-",
CD47tg cells. In certain embodiments, the cells are PCDH11Y", NLGN4Y, B2M,
TRAC-",
CD47tg cells. In certain embodiments, the cells are PCDH11Y-", NLGN4Y-", B2M,
CIITA",
TRB", CD47tg cells. In certain embodiments, the cells are PCDH11Y-", NLGN4Y,
B2M-",
TRB", CD47tg cells. In certain embodiments, the cells are PCDH11Y-", NLGN4Y-",
B2M-",
CHTA", TRAC", TRB", CD47tg cells. In certain embodiments, the cells are
PCDH1117-",
NLGN4Y-", B2M-", TRAC", TRB-", CD47tg cells. In some embodiments, the cells
are
PCDH11171"del/indel, NLGN4Y1deUmdel,
CIITAmdel/uidel , TRACmdel/mdel, CD47tg cells. In
some embodiments, the cells are PCDH 1 IYIndellindel, NLGN4Yindelf111del,
B2mindelfindel, TRAcindel/mdel,
66
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CD47tg cells. In some embodiments, the cells are PCDH1114ndel/indel,
NTGN4yindel/indel,
B2mindel/indel cHTAindeVindel TRBindel/indel, CD47tg cells. In some
embodiments, the cells are
pcpm yindel/indel 1VTGN4y1ndel/indel B2m1nde1finde1 TRBindel/indel,
(71)47tg cells. In some
embodiments, the cells are PCDH11Y
ndel/indel NTGN-41.4nde1/inde1, B2mindel/indel cBTAindel/indel
TRAendelnndel TRBIndel/indel CD47tg cells. In some embodiments, the cells are
PC
Dmiyindel/indel,
NLGN4rndelfindel B2mindel/indel TRAcindel/indel
TRBindelthidel , CD47tg cells. In some embodiments,
the cells are PCDH//Yk""k down, NLGN417kn0 down , B2mkn0ck down , CHTA'''ck
down , TRAcknock down ,
CD47tg cells. In some embodiments, the cells are PCDH11Y1'""0",
NLGN4Ykna'ckcicnvn,
B2m0nock down ,
CIITAknock down TRBknock down, CD47tg cells. In some embodiments, the cells
are
pcp mirnock down , NLGN4Y1'0bock down , B2mkn0ck down , cu TA down ,
TRAcknock down, TRBknock down ,
CD47tg cells. In some embodiments, the cells are PCDH 11Y1"1 ck th'w" ,
NLGN4Y1"0c1"10, B2A41"1 Gic
down , TRAcknock down, CD47tg cells. In some embodiments, the cells are
PCDH//Ylawckd014'n,
NIGN4Yk1ock down, R21 nock down , TRBknock down, CD47tg cells. In some
embodiments, the cells are
PCDHI1Yk" `k d"", NLGN4Yk7"-ck d"", B2Mk0 Gic down, TRACk7wcicdown TRBlawck
down, CD47tg cells.
In some embodiments, the engineered or modified cells described are
pluripotent stem cells (e.g.,
embryonic stem cells or induced pluripotent stem cells), T cells
differentiated from such
pluripotent stem cells or primary T cells. Non-limiting examples of primary T
cells include
CD3+ T cells, CD4+ T cells, CD8+ T cells, naïve T cells, regulatory T (Treg)
cells, non-
regulatory T cells, Thl cells, Th2 cells, Th9 cells, Th17 cells, T-follicular
helper (Tfh) cells,
cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm)
cells, effector
memory T (Tem) cells, effector memory T cells express CD45RA (TEMRA cells),
tissue-
resident memory (Trm) cells, virtual memory T cells, innate memory T cells,
memory stem cell
(Tsc), yo T cells, and any other subtype of T cells. In some embodiments, the
cells are modified
or engineered as compared to a wild-type or control cell, including an
unaltered or unmodified
wild-type cell or control cell. In some embodiments, the wild-type cell or the
control cell is a
starting material. In some embodiments, the starting material is otherwise
modified or
engineered to have altered expression of one or more genes to generate the
engineered cell.
1003451 In some embodiments, a CD47 transgene is inserted into a pre-selected
locus of the
cell. The pre-selected locus can be a safe harbor or target locus. Non-
limiting examples of a
safe harbor or target locus includes a CCR5 gene locus, a CXCR4 gene locus, a
PPP 1R12C gene
locus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene locus, a Rosa
gene locus, an
67
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
F3 (CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1 (CD91)
gene locus, a
HMGB1 gene locus, an ABO gene locus, a REID gene locus, a FUT1 locus, and a
KDM5D gene
locus. In some embodiments, the pre-selected locus is the TRAC locus. In some
embodiments, a
CD47 transgene is inserted into a safe harbor or target locus (e.g., a CCR5
gene locus, a CXCR4
gene locus, a PPP1R12C gene locus, an albumin gene locus, a SHS231 gene locus,
a CLYBL
gene locus, a Rosa gene locus, an F3 (CD142) gene locus, a MICA gene locus, a
MICB gene
locus, a LRP1 (CD91) gene locus, a HN4GB1 gene locus, an ABO gene locus, a RED
gene locus,
a FUT1 locus, and a KDM5D gene locus. In certain embodiments, a CD47 transgene
is inserted
into the B2M locus. In certain embodiments, a CD47 transgene is inserted into
the B2M locus.
In certain embodiments, a CD47 transgene is inserted into the TRAC locus. In
certain
embodiments, a CD47 transgene is inserted into the TRB locus.
[00346] In some instances, expression of a CD47 transgene is controlled by a
constitutive
promoter. In other instances, expression of a CD47 transgene is controlled by
an inducible
promoter. In some embodiments, the promoter is an EFlalpha (EF la) promoter.
In some
embodiments, the promoter a CAG promoter.
[00347] In yet another embodiment, the present disclosure disclosed herein is
directed to
pluripotent stem cells, (e.g., pluripotent stem cells and induced pluripotent
stem cells (iPSCs)), T
cells derived from such pluripotent stem cells (e.g., hypoimmune (HIP) T
cells), and primary T
cells that have reduced expression or lack of expression of one or more Y
chromosome genes
and 1\41IC class I and/or MHC class II human leukocyte antigen molecules and
have reduced
expression or lack of expression of a T-cell receptor (TCR) complex. In some
embodiments, the
cells have reduced or lack of expression of one or more Y chromosome genes and
MHC class I
antigen molecules, MEC class II antigen molecules, and TCR complexes.
[00348] In some embodiments, pluripotent stem cells (e.g., iPSCs),
differentiated cells derived
from such (e.g., T cells, NK cells, cardiac cells, neural cells, cerebral
endothelial cells,
dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid
cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells differentiated
from such), and
primary T cells include a genomic modification or knock down of the PCDH11Y
gene. In some
embodiments, pluripotent stem cells (e.g., iPSCs), differentiated cells
derived from such (e.g., T
cells, NK cells, cardiac cells, neural cells, cerebral endothelial cells,
dopaminergic neurons, glial
progenitor cells, endothelial cells, thyroid cells, hepatocytes, pancreatic
islet cells, and retinal
68
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
pigmented epithelium cells differentiated from such), and primary T cells
include a genomic
modification or knock down of the NLGN4Y gene. In some embodiments,
pluripotent stem cells
(e.g., iPSCs), differentiated cells derived from such (e.g., T cells, NK
cells, cardiac cells, neural
cells, cerebral endothelial cells, dopaminergic neurons, glial progenitor
cells, endothelial cells,
thyroid cells, hepatocytes, pancreatic islet cells, and retinal pigmented
epithelium cells
differentiated from such), and primary T cells include a genomic modification
or knock down of
the B2M gene. In some embodiments, pluripotent stem cells (e.g., iPSCs),
differentiated cells
derived from such (e.g., T cells, NK cells, cardiac cells, neural cells,
cerebral endothelial cells,
dopaminergic neurons, glial progenitor cells, endothelial cells, thyroid
cells, hepatocytes,
pancreatic islet cells, and retinal pigmented epithelium cells differentiated
from such), and
primary T cells include a genomic modification or knock down of the CIITA
gene. In some
embodiments, the cells, including iPSCs and differentiated cells derived from
such pluripotent
stem cells, such as, but not limited to, T cells, NK cells, cardiac cells,
neural cells, cerebral
endothelial cells, dopaminergic neurons, glial progenitor cells, endothelial
cells, thyroid cells,
hepatocytes, pancreatic islet cells, and retinal pigmented epithelium cells,
are PCDH1117-/-,
NLGN4Y, B2M-/- , CIITA cells. In some embodiments, the cells are PCDH 117-/-,
NLGN4Y,
B2M /- cells. In some embodiments, the cells are PCDH Irndel/indel
NLGN4yindeliindel
B2minde1/Inde1 cimindeVendel cells. In some embodiments, the cells are PCDH
lYinciellindel,
ATGN4rndeliindel, 22Apidelfindel cells. In some embodiments, the cells are
PCDH 1 1 Yk"cl d w",
NLGN4yfrnock down , B21knock down CIITAknock down cells. In some embodiments,
the cells are
PCD1-111rn0dk , NLGN4rnock down , B2mknock down cells. In some embodiments,
pluripotent stem
cells (e.g., ESCs or iPSCs), T cells differentiated from such, and primary T
cells include a
genomic modification or knock down of the PCDH11Y gene. In some embodiments,
pluripotent
stem cells (e.g., ESCs or iPSCs), T cells differentiated from such, and
primary T cells include a
genomic modification or knock down of the NLGN4Y gene. In some embodiments,
pluripotent
stem cells (e.g., ESCs or iPSCs), T cells differentiated from such, and
primary T cells include a
genomic modification or knock down of the TRAC gene. In some embodiments,
pluripotent
stem cells (e.g., iPSCs), T cells differentiated from such, and primary T
cells include a genomic
modification or knock down of the TRB gene. In some embodiments, pluripotent
stem cells
(e.g., iPSCs), T cells differentiated from such, and primary T cells include
one or more genomic
modifications or knock downs selected from the group consisting of the B2M,
CIITA and TRAC
69
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
genes. In some embodiments, pluripotent stem cells (e.g., iPSCs), T cells
differentiated from
such, and primary T cells include one or more genomic modifications or knock
downs selected
from the group consisting of the PCDH11Y, NLGN4Y, B2M, CIITA and TRB genes. In
some
embodiments, pluripotent stem cells (e.g., iPSCs), T cells differentiated from
such, and primary
T cells include one or more genomic modifications or knock downs selected from
the group
consisting of the PCDH11Y, NLGN4Y, B2M, CIITA, TRAC and TRB genes. In certain
embodiments, the cells including iPSCs, T cells differentiated from such, and
primary T cells are
PCDH1117', NLGN4Y-', B2111, CIITA, TRAC'cells. In certain embodiments, the
cells
including iPSCs, T cells differentiated from such, and primary T cells are
PCDH1117-/-, NLGN4Y-
/-, B2M- TRAC-/-cells. In certain embodiments, the cells including iPSCs, T
cells differentiated
from such, and primary T cells are PCDH11Y-/-, NLGN4Y, B2A1-/- , CIITA,
TRB'cells. In
certain embodiments, the cells including iPSCs, T cells differentiated from
such, and primary T
cells are PCDH1117-/ NLGN4Y, B211/1-/ TRB"cells. In certain embodiments, the
cells
including iPSCs, T cells differentiated from such, and primary T cells are
PCDH1JY, NLGN4Y-
/-, B2M, CIITA, TRAC- TRIP/ cells. In certain embodiments, the cells including
iPSCs, T
cells differentiated from such, and primary T cells are PCDH11Y, NLGN4Y,
B211,1-/-, TRACY-,
TRB-/-cells. In some embodiments, the cells including iPSCs, T cells
differentiated from such,
and primary T cells are PCDH11Yindel/indel, NLGN4yinde1/inde1, B2mindel/mdel
CHTAindel/nadel
TRACinaellindel cells. In some embodiments, the cells including iPSCs, T cells
differentiated from
such, and primary T cells are PCDH 1 lYinde', NLGN4rndel/indel, B2mindel/indel
TRAcindel/indel
cells. In some embodiments, the cells including iPSCs, T cells differentiated
from such, and
primary T cells are PCDH1 lYindeui"del, NLGN4Y1ndelfinde1, B2Mindeilindel,
TRBindelel
cells. In some embodiments, the cells including iPSCs, T cells differentiated
from such, and
primary T cells are PCDH11YindeL/indel, NLGN4Yindel/indel, B2mindeL/indel
TRBindeLlndel cells. In some
embodiments, the cells including iPSCs, T cells differentiated from such, and
primary T cells are
PCDH11Yindellindel NLGN4Yindel/indel, BNI/Tindel/indel CIITAm0d indel
TRACindel/indel TRBindel/indel cells.
In some embodiments, the cells including iPSCs, T cells differentiated from
such, and primary T
cells are PCDH//Pndel/indel, NLGN-Iyindel/indel, B21 ndel/indel,
TRACindel/indel TRBindekindel cells. In
some embodiments, the cells including ESCs, iPSCs, T cells differentiated from
such, and
primary T cells are PCDH11Yk" c1c d w", NLGN4Y
knock down, B2mkn0ck down , cu TA
d"", TRAClawck
down cells. In some embodiments, the cells including iPSCs, T cells
differentiated from such, and
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
primary T cells are PCDH lYk"ck down, NLGN4Y
knock down , B2mknock down , CHTAknock down , TRBknock
down cells. In some embodiments, the cells including ESCs, iPSCs, T cells
differentiated from
such, and primary T cells are /,(7Ty_ yknock down, NTGN4ykn0ck down, B2mknock
down, lliAknock down ,
TRAcknock clown TRI3kn0 cd wfl cells. In some embodiments, the cells including
ESCs, iPSCs, T cells
differentiated from such, and primary T cells are PCDH1 1Y
knock down , NLGN4ykn0ck down , B2mknock
down , TRAcknock down cells. In some embodiments, the cells including iPSCs, T
cells differentiated
from such, and primary T cells are PCDH//Ykiwck down, NLGN4Ykn0ck down,
B211/11 106* cabwn TRBknock
down cells. In some embodiments, the cells including ESCs, iPSCs, T cells
differentiated from
such, and primary T cells are PCDH 1 lYkn ck d wn, NLGN4Ykn ck 47 14'n ,
B21knock down TRACknock down ,
TRBI 1 d cd wfl cells. In some embodiments, the modified cells described are
pluripotent stem cells,
induced pluripotent stem cells, T cells differentiated from such pluripotent
stem cells and
induced pluripotent stem cells, or primary T cells. Non-limiting examples of
primary T cells
include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T
(Treg) cells, non-
regulatory T cells, Thl cells, Th2 cells, Th9 cells, Th17 cells, T-follicular
helper (Tfh) cells,
cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tem)
cells, effector
memory T (Tem) cells, effector memory T cells express CD45RA (TEMRA cells),
tissue-
resident memory (Trm) cells, virtual memory T cells, innate memory T cells,
memory stem cell
(Tsc), y6 T cells, and any other subtype of T cells.
1003491 In some embodiments, the cells are modified or engineered as compared
to a wild-type
or control cell, including an unaltered or unmodified wild-type cell or
control cell. In some
embodiments, the wild-type cell or the control cell is a starting material. In
some embodiments,
the starting material is otherwise modified or engineered to have reduced or
lack of expression of
one or more Y chromosome genes, including but not limited to, PCDH11Y and/or
NLGN4Y.
Reduction of PCDHI lY and/or NLGN4Y expression can be accomplished, for
example, by
targeting the PCDHI IY and NLGN4Y genes directly; and/or by targeting
components that are
critical for their transcription, translation, or protein stability.
[00350] Cells of the present disclosure exhibit reduced or lack of expression
of MHC class I
antigen molecules, MEC class II antigen molecules, and/or TCR complexes.
Reduction of one or
more MHC class I and/or class II 1-ILA molecules expression can be
accomplished, for example,
by one or more of the following: (1) targeting the polymorphic 1-ILA alleles
(HLA-A, HLA-B,
HLA-C) and MHC-II genes directly; (2) removal of B2M, which will prevent
surface trafficking
71
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
of all MFIC-I molecules; (3) removal of CIITA, which will prevent surface
trafficking of all
MtIC-II molecules; and/or (4) deletion of components of the MHC enhanceosomes,
such as
LRC5, RFX5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and
CIITA
that are critical for I-ILA expression.
1003511 In some embodiments, HLA expression is interfered with by targeting
individual HLAs
(e.g., knocking out, knocking down, or reducing expression of HLA-A, HLA-B,
HLA-C, HLA-
DP, HLA-DQ, and/or HLA-DR), targeting transcriptional regulators of HLA
expression (e.g.,
knocking out, knocking down, or reducing expression of NLRC5, CIITA, RFX5,
RFXAP,
RFXANK, NFY-A, NFY-B, NFY-C and/or IRF-1), blocking surface trafficking of
MFIC class I
molecules (e.g., knocking out, knocking down, or reducing expression of B2M
and/or TAP1),
and/or targeting with HLA-Razor (see, e.g., W02016183041).
1003521 In some embodiments, the cells disclosed herein including, but not
limited to,
pluripotent stem cells, induced pluripotent stem cells, differentiated cells
derived from such stem
cells, and primary T cells do not express one or more human leukocyte antigen
molecules (e.g.,
HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and/or HLA-DR) corresponding to MHC-I
molecules and/or molecules and arc thus characterized as being
hypoimmunogcnic. For
example, in certain embodiments, the pluripotent stem cells and induced
pluripotent stem cells
disclosed have been modified such that the stem cell or a differentiated stem
cell prepared
therefrom do not express or exhibit reduced expression of one or more of the
following MI-IC-I
molecules: HLA-A, HLA-B and ERA-C. In some embodiments, one or more of HLA-A,
fiLA-B
and HLA-C may be "knocked-out" of a cell. A cell that has a knocked-out HLA-A
gene, HLA-B
gene, and/or LILA-C gene may exhibit reduced or eliminated expression of each
knocked-out
gene.
1003531 In some embodiments, guide RNAs, shRNAs, siRNAs, or miRNAs that allow
simultaneous deletion of all MFIC class I alleles by targeting a conserved
region in the HLA
genes are identified as HLA Razors. In some embodiments, the gRNAs are part of
a CRISPR
system. In alternative embodiments, the gRNAs are part of a TALEN system. In
some
embodiments, an HLA Razor targeting an identified conserved region in HLAs is
described in
W02016183041. In some embodiments, multiple HLA Razors targeting identified
conserved
regions are utilized. It is generally understood that any guide, siRNA, shRNA,
or miRNA
molecule that targets a conserved region in HLAs can act as an HLA Razor.
72
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1003541 Methods provided are useful for inactivation or ablation of MEC class
I molecule
expression and/or MI-1C class II molecule expression in cells such as but not
limited to
pluripotent stem cells, differentiated cells, and primary T cells. In some
embodiments, genome
editing technologies utilizing rare-cutting endonucleases (e.g., the
CRISPR/Cas, TALEN, zinc
finger nuclease, meganuclease, and homing endonuclease systems) are also used
to reduce or
eliminate expression of genes involved in an immune response (e.g., by
deleting genomic DNA
of genes involved in an immune response or by insertions of genomic DNA into
such genes,
such that gene expression is impacted) in cells. In certain embodiments,
genome editing
technologies or other gene modulation technologies are used to insert
tolerance-inducing factors
in human cells, rendering them and the differentiated cells prepared therefrom
hypoimmunogenic
cells. As such, the engineered and/or hypoimmunogenic cells have reduced or
eliminated
expression of MHC I molecule and/or MEC II molecule expression. In some
embodiments, the
cells are nonimmunogenic (e.g., do not induce an innate and/or an adaptive
immune response) in
a recipient subject.
1003551 In some embodiments, the cell includes a modification to increase
expression of CD47
and one or more factors selected from the group consisting of DUX4, CD24,
CD27, CD46,
CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, lD01, CTLA4-
Ig,
Cl-Inhibitor, 1L-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, and Serpinb9.
1003561 In some embodiments, the cell comprises a genomic modification of one
or more target
polynucleotide sequences that regulate the expression of either MEIC class I
molecules, MHC
class II molecules, or MHC class I and MHC class II molecules. In some
embodiments, a
genetic editing system is used to modify one or more target polynucleotide
sequences. In some
embodiments, an RNAi system is used to knock down expression of one or more
target
polynucleotide sequences. In some embodiments, the targeted polynucleotide
sequence is one or
more selected from the group including B2M, CIITA, and NLRC5. In some
embodiments, the
cell comprises a genetic editing modification to the B2M gene. In some
embodiments, the cell
comprises a genetic editing modification to the CIITA gene. In some
embodiments, the cell
comprises a genetic editing modification to the NLRC5 gene. In some
embodiments, the cell
comprises genetic editing modifications to the B2M and CIITA genes. In some
embodiments, the
cell comprises genetic editing modifications to the B2M and NLRC5 genes. In
some
embodiments, the cell comprises genetic editing modifications to the CIITA and
NLRC5 genes.
73
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
In numerous embodiments, the cell comprises genetic editing modifications to
the B2M, CIITA
and NLRC5 genes. In certain embodiments, the genome of the cell has been
altered to reduce or
delete critical components of HLA expression. In some embodiments, the cells
are modified or
engineered as compared to a wild-type or control cell, including an unaltered
or unmodified
wild-type cell or control cell. In some embodiments, the wild-type cell or the
control cell is a
starting material. In some embodiments, the starting material is otherwise
modified or
engineered to have altered expression of one or more genes to generate the
engineered cell.
1003571 In some embodiments, the present disclosure provides a cell (e.g.,
stem cell, induced
pluripotent stem cell, differentiated cell such as a cardiac cell, neural
cell, cerebral endothelial
cell, dopaminergic neuron, glial progenitor cell, endothelial cell, thyroid
cell, hepatocyte,
pancreatic islet cell, or retinal pigmented epithelium cell, hematopoietic
stem cell, primary NK
cell, CAR-NK cell, primary T cell or CAR-T cell) or population thereof
comprising a genome in
which a gene has been edited to delete a contiguous stretch of genomic DNA,
thereby reducing
or eliminating surface expression of MEW class I molecules in the cell or
population thereof. In
certain embodiments, the present disclosure provides a cell (e.g., stem cell,
induced pluripotent
stem cell, differentiated cell such as a cardiac cell, neural cell, cerebral
endothelial cell,
dopaminergic neuron, glial progenitor cell, endothelial cell, thyroid cell,
hepatocyte, pancreatic
islet cell, or retinal pigmented epithelium cell, hematopoietic stem cell,
primary NK cell, CAR-
NK cell, primary T cell or CAR-T cell) or population thereof comprising a
genome in which a
gene has been edited to delete a contiguous stretch of genomic DNA, thereby
reducing or
eliminating surface expression of MEW class II molecules in the cell or
population thereof In
numerous embodiments, the present disclosure provides a cell (e.g., stem cell,
induced
pluripotent stem cell, differentiated cell such as a cardiac cell, neural
cell, cerebral endothelial
cell, dopaminergic neuron, glial progenitor cell, endothelial cell, thyroid
cell, hepatocyte,
pancreatic islet cell, or retinal pigmented epithelium cell, hematopoietic
stem cell, primary NK
cell, CAR-NK cell, primary T cell or CAR-T cell) or population thereof
comprising a genome in
which one or more genes has been edited to delete a contiguous stretch of
genomic DNA,
thereby reducing or eliminating surface expression of MEC class I and II
molecules in the cell or
population thereof.
1003581 In certain embodiments, the expression of one or more MFIC I molecules
and/or MHC
II molecules (including one or more MHC class I and/or class II HLA molecules)
is modulated
74
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
by targeting and deleting a contiguous stretch of genomic DNA, thereby
reducing or eliminating
expression of a target gene selected from the group consisting of B2M, CI1TA,
and NLRC5. In
some embodiments, described herein are genetically edited cells (e.g.,
modified human cells)
comprising exogenous CD47 proteins and inactivated or modified CIITA gene
sequences, and in
some instances, additional gene modifications that inactivate or modify B2M
gene sequences. In
some embodiments, described herein are genetically edited cells comprising
exogenous CD47
proteins and inactivated or modified CIITA gene sequences, and in some
instances, additional
gene modifications that inactivate or modify NLRC5 gene sequences. In some
embodiments,
described herein are genetically edited cells comprising exogenous CD47
proteins and
inactivated or modified B2M gene sequences, and in some instances, additional
gene
modifications that inactivate or modify NLRC5 gene sequences. In some
embodiments,
described herein are genetically edited cells comprising exogenous CD47
proteins and
inactivated or modified B2M gene sequences, and in some instances, additional
gene
modifications that inactivate or modify CIITA gene sequences and NLRC5 gene
sequences.
1003591 Provided herein are cells exhibiting a modification of one or more
targeted
polynucleotide sequences that regulates the expression of any one of the
following: (a) MHC I
antigen molecules, (b) MHC II antigen molecules, (c) TCR complexes, (d) both
MHC I and II
antigen molecules, and (e) MHC I and II antigen molecules and TCR complexes.
In certain
embodiments, the modification includes increasing expression of CD47. In some
embodiments,
the cells include an exogenous or recombinant CD47 polypeptide. In certain
embodiments, the
modification includes expression of a chimeric antigen receptor. In some
embodiments, the cells
comprise an exogenous or recombinant chimeric antigen receptor polypeptide.
1003601 In some embodiments, the cell includes a genomic modification of one
or more targeted
polynucleotide sequences that regulates the expression of one or more MHC I
antigen molecules,
MHC II antigen molecules and/or TCR complexes. In some embodiments, a genetic
editing
system is used to modify one or more targeted polynucleotide sequences. In
some embodiments,
the polynucleotide sequence targets one or more genes selected from the group
consisting of
B2M, CIITA, TRAC, and TRB. In certain embodiments, the genome of a T cell
(e.g., a T cell
differentiated from hypoimmunogenic iPSCs and a primary T cell) has been
altered to reduce or
delete critical components of HLA and TCR expression, e.g., HLA-A antigen, HLA-
B antigen,
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
HLA-C antigen, HLA-DP antigen, HLA-DQ antigen, HLA-DR antigens, TCR-alpha and
TCR-
beta.
[00361] In some embodiments, the present disclosure provides a cell or
population thereof
comprising a genome in which a gene has been edited to delete a contiguous
stretch of genomic
DNA, thereby reducing or eliminating surface expression of MHC class I
molecules in the cell or
population thereof. In certain embodiments, the present disclosure provides a
cell or population
thereof comprising a genome in which a gene has been edited to delete a
contiguous stretch of
genomic DNA, thereby reducing or eliminating surface expression of MHC class
11 molecules in
the cell or population thereof. In certain embodiments, the present disclosure
provides a cell or
population thereof comprising a genome in which a gene has been edited to
delete a contiguous
stretch of genomic DNA, thereby reducing or eliminating surface expression of
TCR molecules
in the cell or population thereof. In numerous embodiments, the present
disclosure provides a
cell or population thereof comprising a genome in which one or more genes has
been edited to
delete a contiguous stretch of genomic DNA, thereby reducing or eliminating
surface expression
of one or more MHC class I and II molecules and TCR complex molecules in the
cell or
population thereof.
[00362] In some embodiments, the cells and methods described herein include
genomically
editing human cells to cleave CIITA gene sequences as well as editing the
genome of such cells
to alter one or more additional target polynucleotide sequences such as, but
not limited to,
PCDH11Y, NLGN4Y, B2M TRAC, and TRB. In some embodiments, the cells and methods

described herein include genomically editing human cells to cleave B2M gene
sequences as well
as editing the genome of such cells to alter one or more additional target
polynucleotide
sequences such as, but not limited to, PCDH11Y, NLGN4Y, CIITA, TRAC, and TRB.
In some
embodiments, the cells and methods described herein include genomically
editing human cells to
cleave TRAC gene sequences as well as editing the genome of such cells to
alter one or more
additional target polynucleotide sequences such as, but not limited to,
PCDH11Y, NLGN4Y,
B2M, CIITA, and TRB. In some embodiments, the cells and methods described
herein include
genomically editing human cells to cleave TRB gene sequences as well as
editing the genome of
such cells to alter one or more additional target polynucleotide sequences
such as, but not limited
to, PCDH11Y, NLGN4Y, B2M, CIITA, and TRAC.
76
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1003631 Provided herein are hypoimmunogenic stem cells comprising reduced
expression of
PCDH11Y and/or NLGN4Y and HLA-A, HLA-C, CIITA, TCR-alpha, and
TCR-beta
relative to a wild-type stem cell, the hypoimmunogenic stem cell further
comprising a set of
exogenous polynucleotides comprising a first exogenous polynucleotide encoding
CD47 and a
second exogenous polynucleotide encoding a chimeric antigen receptor (CAR),
wherein the first
and/or second exogenous polynucleotides are inserted into a specific locus of
at least one allele
of the cell. Also provided herein are hypoimmunogenic piimaiy T cells
including any subtype of
primary T cells comprising reduced expression of PCDH11Y and/or NLGN4Y and HLA-
A,
HLA-B, HLA-C, CIITA, TCR-alpha, and TCR-beta relative to a wild-type primary T
cell, the
hypoimmunogenic stem cell further comprising a set of exogenous
polynucleotides comprising a
first exogenous polynucleotide encoding CD47 and a second exogenous
polynucleotide encoding
a chimeric antigen receptor (CAR), wherein the first and/or second exogenous
polynucleotides
are inserted into a specific locus of at least one allele of the cell. Further
provided herein are
hypoimmunogenic T cells differentiated from hypoimmunogenic induced
pluripotent stem cells
comprising reduced expression of PCDHI1Y and/or NLGN4Y and HLA-A, HLA-B, HLA-
C,
CIITA, TCR-alpha, and TCR-beta relative to a wild-type primary T cell, the
hypoimmunogcnic
stem cell further comprising a set of exogenous polynucleotides comprising a
first exogenous
polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a
chimeric
antigen receptor (CAR), wherein the first and/or second exogenous
polynucleotides are inserted
into a specific locus of at least one allele of the cell.
1003641 In some embodiments, the population of engineered cells described
evades NK cell
mediated cytotoxicity upon administration to a patient. In some embodiments,
the population of
engineered cells evades NK cell mediated cytotoxicity by one or more
subpopulations of NK
cells. In some embodiments, the population of engineered cells is protected
from cell lysis by
NK cells, including immature and/or mature NK cells upon administration to a
patient. In some
embodiments, the population of engineered cells evades macrophage engulfment
upon
administration to a patient. In some embodiments, the population of engineered
cells does not
induce an innate and/or an adaptive immune response to the cell upon
administration to a patient.
1003651 In some embodiments, the cells described herein comprise a safety
switch. The term
"safety switch" used herein refers to a system for controlling the expression
of a gene or protein
of interest that, when downregulated or upregulated, leads to clearance or
death of the cell, e.g.,
77
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
through recognition by the host's immune system. A safety switch can be
designed to be
triggered by an exogenous molecule in case of an adverse clinical event. A
safety switch can be
engineered by regulating the expression on the DNA, RNA and protein levels. A
safety switch
includes a protein or molecule that allows for the control of cellular
activity in response to an
adverse event. In one embodiment, the safety switch is a "kill switch" that is
expressed in an
inactive state and is fatal to a cell expressing the safety switch upon
activation of the switch by a
selective, externally provided agent. In one embodiment, the safety switch
gene is cis-acting in
relation to the gene of interest in a construct. Activation of the safety
switch causes the cell to
kill solely itself or itself and neighboring cells through apoptosis or
necrosis. In some
embodiments, the cells described herein, e.g., stem cells, induced pluripotent
stem cells,
hematopoietic stem cells, primary cells, or differentiated cell, including,
but not limited to, T
cells, CAR-T cells, NK cells, and/or CAR-NK cells, comprise a safety switch.
1003661 In some embodiments, the safety switch comprises a therapeutic agent
that inhibits or
blocks the interaction of CD47 and SIRPa. In some aspects, the CD47-SIRPa
blockade agent is
an agent that neutralizes, blocks, antagonizes, or interferes with the cell
surface expression of
CD47, SIRPa, or both. In some embodiments, the CD47-SIRPa blockade agent
inhibits or
blocks the interaction of CD47, SIRPa or both. In some embodiments, a CD47-
SIRPot blockade
agent (e.g., a CD47-SIRPa blocking, inhibiting, reducing, antagonizing,
neutralizing, or
interfering agent) comprises an agent selected from from a group that includes
an antibody or
fragment thereof that binds CD47, a bispecific antibody that binds CD47, an
immunocytokine
fusion protein that bind CD47, a CD47 containing fusion protein, an antibody
or fragment
thereof that binds SIRPa, a bispecific antibody that binds SIRPa, an
immunocytokine fusion
protein that bind SIRPa, an SIRPa containing fusion protein, and a combination
thereof.
1003671 In some embodiments, the cells described herein comprise a "suicide
gene" (or "suicide
switch-). The suicide gene can cause the death of the hypoimmunogenic cells
should they grow
and divide in an undesired manner. The suicide gene ablation approach includes
a suicide gene in
a gene transfer vector encoding a protein that results in cell killing only
when activated by a
specific compound. A suicide gene can encode an enzyme that selectively
converts a nontoxic
compound into highly toxic metabolites. In some embodiments, the cells
described herein, e.g.,
stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary
cells, or
78
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
differentiated cell, including, but not limited to, T cells, CAR-T cells, NK
cells, and/or CAR-NK
cells, comprise a suicide gene.
1003681 In some embodiments, the population of engineered cells described
elicits a reduced
level of immune activation or no immune activation upon administration to a
recipient subject. In
some embodiments, the cells elicit a reduced level of systemic TH1 activation
or no systemic
TH1 activation in a recipient subject. In some embodiments, the cells elicit a
reduced level of
immune activation of peripheral blood mononuclear cells (PBMCs) or no immune
activation of
PBMCs in a recipient subject. In some embodiments, the cells elicit a reduced
level of donor-
specific IgG antibodies or no donor specific IgG antibodies against the cells
upon administration
to a recipient subject. In some embodiments, the cells elicit a reduced level
of IgM and IgG
antibody production or no IgM and IgG antibody production against the cells in
a recipient
subject. In some embodiments, the cells elicit a reduced level of cytotoxic T
cell killing of the
cells upon administration to a recipient subject.
B. CIITA
[00369] In some embodiments, the technologies disclosed herein modulate (e.g.,
reduce or
eliminate) the expression of MEW II genes by targeting and modulating (e.g.,
reducing or
eliminating) Class II transactivator (CIITA) expression. In some embodiments,
the modulation
occurs using a gene editing system (e.g. CRISPR/Cas system).
1003701 CIITA is a member of the LR or nucleotide binding domain (NBD) leucine-
rich repeat
(LRR) family of proteins and regulates the transcription of MTIC II by
associating with the M_HC
enhanceosome.
[00371] In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of CIITA. In some embodiments, the target polynucleotide sequence is a
homolog of
CIITA. In some embodiments, the target polynucleotide sequence is an ortholog
of CIITA.
[00372] In some embodiments, reduced or eliminated expression of CIITA reduces
or
eliminates expression of one or more of the following MHC class II molecules
are 1-11,A-DP,
HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.
[00373] In some embodiments, the cells described herein comprise gene
modifications at the
gene locus encoding the CIITA protein. In other words, the cells comprise a
genetic
modification at the CIITA locus. In some instances, the nucleotide sequence
encoding the
79
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CIITA protein is set forth in RefSeq. No. NM 000246.4 and NCBI Genbank No.
U18259. In
some instances, the CIITA gene locus is described in NCBI Gene ID No. 4261. In
certain cases,
the amino acid sequence of CIITA is depicted as NCBI GenBank No. AAA88861.1.
Additional
descriptions of the CIITA protein and gene locus can be found in Uniprot No.
P33076, HGNC
Ref No. 7067, and OMIM Ref No. 600005.
[00374] In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the CIITA gene. In some embodiments,
the genetic
modification targeting the CIITA gene by the rare-cutting endonuclease
comprises a Cos protein
or a polynucleotide encoding a Cos protein, and at least one guide ribonucleic
acid sequence for
specifically targeting the CIITA gene. In some embodiments, the at least one
guide ribonucleic
acid sequence for specifically targeting the CIITA gene is selected from the
group consisting of
SEQ ID NOS:5184-36352 of Table 12 of W02016183041, which is herein
incorporated by
reference. In some embodiments, the cell has a reduced ability to induce an
innate and/or an
adaptive immune response in a recipient subject. In some embodiments, an
exogenous nucleic
acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen
receptor, CD47, or
another tolerogenic factor disclosed herein) is inserted at the CIITA gene.
[00375] In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a knock out of CIITA expression, such that the cells are CIITA. In
some
embodiments, the engineered and/or hypoimmunogenic cells outlined herein
introduce an indel
into the CIITA gene locus, such that the cells are CIITA"'"'. In some
embodiments, the
engineered and/or hypoimmunogenic cells outlined herein comprise knock down of
CIITA
expression, such that the cells are CHTAknock down .
[00376] Assays to test whether the CIITA gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the CIITA
gene by PCR and
the reduction of HLA-II expression can be assays by FACS analysis. In another
embodiment,
CIITA protein expression is detected using a Western blot of cells lysates
probed with antibodies
to the CIITA protein. In another embodiment, reverse transcriptase polymerase
chain reactions
(RT-PCR) are used to confirm the presence of the inactivating genetic
modification.
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
C. B2M
1003771 In some embodiments, the technologies disclosed herein modulate (e.g.,
reduce or
eliminate) the expression of MEIC-I genes by targeting and modulating (e.g.,
reducing or
eliminating) expression of the accessory chain B2M. In some embodiments, the
modulation
occurs using a gene editing system (e.g. CRISPR/Cas system).
[00378] By modulating (e.g., reducing or deleting) expression of B2M, surface
trafficking of
MEIC-I molecules is blocked and the cell rendered hypoimmunogenic. In some
embodiments,
the cell has a reduced ability to induce an innate and/or an adaptive immune
response in a
recipient subject.
1003791 In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of B2M. In some embodiments, the target polynucleotide sequence is a
homolog of B2M.
In some embodiments, the target polynucleotide sequence is an ortholog of B2M.
1003801 In some embodiments, decreased or eliminated expression of B2M reduces
or
eliminates expression of one or more of the following MHC I molecules: HLA-A,
HLA-B, and
HLA-C.
[00381] In some embodiments, the cells described herein comprise gene
modifications at the
gene locus encoding the B2M protein. In other words, the cells comprise a
genetic modification
at the B2M locus. In some instances, the nucleotide sequence encoding the B2M
protein is set
forth in RefSeq. No. NM 004048.4 and Genbank No. AB021288.1. In some
instances, the B2M
gene locus is described in NCBI Gene ID No. 567. In certain cases, the amino
acid sequence of
B2M is depicted as NCBI GenBank No. BAA35182.1. Additional descriptions of the
B2M
protein and gene locus can be found in Uniprot No. P61769, HGNC Ref. No. 914,
and OMINI
Ref No. 109700.
[00382] In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the B2M gene. In some embodiments,
the genetic
modification targeting the B2M gene by the rare-cutting endonuclease comprises
a Cas protein
or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic
acid sequence for
specifically targeting the B2M gene. In some embodiments, the at least one
guide ribonucleic
acid sequence for specifically targeting the B2M gene is selected from the
group consisting of
SEQ ID NOS:81240-85644 of Table 15 of W02016183041, which is herein
incorporated by
reference. In some embodiments, an exogenous nucleic acid encoding a
polypeptide as disclosed
81
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor
disclosed herein) is
inserted at the B2M gene.
1003831 Assays to test whether the B2M gene has been inactivated are known and
described
herein. In some embodiments, the resulting genetic modification of the B2M
gene by PCR and
the reduction of EILA-I expression can be assays by FACS analysis. In another
embodiment,
B2M protein expression is detected using a Western blot of cells lysates
probed with antibodies
to the B21VI protein. In another embodiment, reverse transcriptase polymerase
chain reactions
(RT-PCR) are used to confirm the presence of the inactivating genetic
modification.
1003841 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise knock out of B2M expression, such that the cells are B211/1. In some
embodiments,
the engineered and/or hypoimmunogenic cells outlined herein introduce an indel
into the B2M
gene locus, such that the cells are B2Mindel/mdel In some embodiments, the
engineered and/or
hypoimmunogenic cells outlined herein comprise knock down of B2M expression,
such that the
cells are B2/1/0Gicd0"'".
D. NLRC5
1003851 In certain embodiments, the technologies disclosed herein modulate
(e.g., reduce or
eliminate) the expression of MTIC-I genes by targeting and modulating (e.g.,
reducing or
eliminating) expression of the NLR family, CARD domain containing
5/NOD27/CLR16.1
(NLRC5). In some embodiments, the modulation occurs using a gene editing
system (e.g.
CRISPR/Cas system).
1003861 NLRC5 is a critical regulator of MHC-I-mediated immune responses and,
similar to
CIITA, NLRC5 is highly inducible by IFN-7 and can translocate into the
nucleus. NLRC5
activates the promoters of MHC-I genes and induces the transcription of MHC-I
as well as
related genes involved in MEIC-I antigen presentation.
1003871 In some embodiments, the target polynucleotide sequence is a variant
of NLRC5. In
some embodiments, the target polynucleotide sequence is a homolog of NLRC5. In
some
embodiments, the target polynucleotide sequence is an ortholog of NLRC5.
1003881 In some embodiments, decreased or eliminated expression of NLRC5
reduces or
eliminates expression of one or more of the following MHC I molecules ¨ HLA-A,
HLA-B, and
HLA-C.
82
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00389] In some embodiments, the cells outlined herein comprise a genetic
modification
targeting the NLRC5 gene. In some embodiments, the genetic modification
targeting the
NLRC5 gene by the rare-cutting endonuclease comprises a Cas protein or a
polynucleotide
encoding a Cas protein, and at least one guide ribonucleic acid sequence for
specifically
targeting the NLRC5 gene. In some embodiments, the at least one guide
ribonucleic acid
sequence for specifically targeting the NLRC5 gene is selected from the group
consisting of SEQ
ID NOS.36353-81239 of Appendix 3 or Table 14 of W02016183041, the disclosure
is
incorporated by reference in its entirety.
[00390] Assays to test whether the NLRC5 gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the NLRC5
gene by PCR
and the reduction of HLA-I expression can be assays by FACS analysis. In
another embodiment,
NLRC5 protein expression is detected using a Western blot of cells lysates
probed with
antibodies to the NLRC5 protein. In another embodiment, reverse transcriptase
polymerase
chain reactions (RT-PCR) are used to confirm the presence of the inactivating
genetic
modification.
[00391] In some embodiments, the engineered and/or hypoimmunogcnic cells
outlined herein
comprise knock out of NLRC5 expression, such that the cells are NLRC5. In some

embodiments, the engineered and/or hypoimmunogenic cells outlined herein
introduce an indel
into the NLRC5 gene locus, such that the cells are N112(75`"de1thide1 . In
some embodiments, the
engineered and/or hypoimmunogenic cells outlined herein comprise knock down of
NLRC5
expression, such that the cells are NLRC5' 'd w"
E. TRAC
1003921 In certain embodiments, the technologies disclosed herein modulate
(e.g., reduce or
eliminate) the expression of TCR genes including the TRAC gene by targeting
and modulating
(e.g., reducing or eliminating) expression of the constant region of the T
cell receptor alpha
chain. In some embodiments, the modulation occurs using a gene editing system
(e.g.
CRISPR/Cas system).
[00393] By modulating (e.g., reducing or deleting) expression of 1RAC, surface
trafficking of
TCR molecules is blocked. In some embodiments, the cell also has a reduced
ability to induce
an innate and/or an adaptive immune response in a recipient subject.
83
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1003941 In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of TRAC. In some embodiments, the target polynucleotide sequence is a
homolog of
TRAC. In some embodiments, the target polynucleotide sequence is an ortholog
of TRAC.
1003951 In some embodiments, decreased or eliminated expression of TRAC
reduces or
eliminates TCR surface expression.
1003961 In some embodiments, the cells, such as, but not limited to,
pluripotent stem cells,
induced pluripotent stem cells, T cells differentiated from induced
pluripotent stein cells, primary
T cells, and cells derived from primary T cells comprise gene modifications at
the gene locus
encoding the TRAC protein. In other words, the cells comprise a genetic
modification at the
TRAC locus. In some instances, the nucleotide sequence encoding the TRAC
protein is set forth
in Genbank No. X02592.1. In some instances, the TRAC gene locus is described
in RefSeq. No.
NG 001332.3 and NCBI Gene ID No. 28755. In certain cases, the amino acid
sequence of
TRAC is depicted as Uniprot No. P01848. Additional descriptions of the TRAC
protein and gene
locus can be found in Uniprot No. P01848, HGNC Ref No. 12029, and OMIM Ref.
No. 186880.
1003971 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the TRAC gene. In some embodiments,
the genetic
modification targeting the TRAC gene by the rare-cutting endonuclease
comprises a Cas protein
or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic
acid sequence for
specifically targeting the TRAC gene. In some embodiments, the at least one
guide ribonucleic
acid sequence for specifically targeting the TRAC gene is selected from the
group consisting of
SEQ ID NOS:532-609 and 9102-9797 of US20160348073, which is herein
incorporated by
reference.
1003981 Assays to test whether the TRAC gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the TRAC
gene by PCR and
the reduction of TCR expression can be assays by FACS analysis. In another
embodiment,
TRAC protein expression is detected using a Western blot of cells lysates
probed with antibodies
to the TRAC protein. In another embodiment, reverse transcriptase polymerase
chain reactions
(RT-PCR) are used to confirm the presence of the inactivating genetic
modification.
1003991 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise knock out of TRAC expression, such that the cells are TRAC- In some
embodiments,
the engineered and/or hypoimmunogenic cells outlined herein introduce an indel
into the TRAC
84
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
gene locus, such that the cells are TRACIndeVindel . In some embodiments, the
engineered and/or
hypoimmunogenic cells outlined herein comprise knock down of TRAC expression,
such that
the cells are 1R4Ckn ck down .
F. TRB
1004001 In certain embodiments, the technologies disclosed herein modulate
(e.g., reduce or
eliminate) the expression of TCR genes including the gene encoding T cell
antigen receptor, beta
chain (e.g., the TRB, TRBC, or TCRB gene) by targeting and modulating (e.g.,
reducing or
eliminating) expression of the constant region of the T cell receptor beta
chain. In some
embodiments, the modulation occurs using a gene editing system (e.g.
CRISPR/Cas system).
[00401] By modulating (e.g., reducing or deleting) expression of TRB, surface
trafficking of
TCR molecules is blocked. In some embodiments, the cell also has a reduced
ability to induce an
innate and/or an adaptive immune response in a recipient subject.
1004021 In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of TRB. In some embodiments, the target polynucleotide sequence is a
homolog of TRB.
In some embodiments, the target polynucleotide sequence is an ortholog of TRB.
[00403] In some embodiments, decreased or eliminated expression of TRB reduces
or
eliminates TCR surface expression.
[00404] In some embodiments, the cells, such as, but not limited to,
pluripotent stem cells,
induced pluripotent stem cells, T cells differentiated from induced
pluripotent stem cells, primary
T cells, and cells derived from primary T cells comprise gene modifications at
the gene locus
encoding the TRB protein. In other words, the cells comprise a genetic
modification at the TRB
gene locus. In some instances, the nucleotide sequence encoding the TRB
protein is set forth in
UniProt No. PODSE2. In some instances, the TRB gene locus is described in
RefSeq. No.
NG 001333.2 and NCBI Gene ID No. 6957. In certain cases, the amino acid
sequence of TRB
is depicted as Uniprot No. P01848. Additional descriptions of the TRB protein
and gene locus
can be found in GenBank No. L36092.2, Uniprot No. PODSE2, and HGNC Ref. No.
12155.
1004051 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the TRB gene. In some embodiments,
the genetic
modification targeting the TRB gene by the rare-cutting endonuclease comprises
a Cas protein or
a polynucleotide encoding a Cas protein, and at least one guide ribonucleic
acid sequence for
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
specifically targeting the TRB gene. In some embodiments, the at least one
guide ribonucleic
acid sequence for specifically targeting the TRB gene is selected from the
group consisting of
SEQ ID NOS:610-765 and 9798-10532 of US20160348073, which is herein
incorporated by
reference.
[00406] Assays to test whether the TRB gene has been inactivated are known and
described
herein. In some embodiments, the resulting genetic modification of the TRB
gene by PCR and
the reduction of TCR expression can be assays by FACS analysis. In another
embodiment, TRB
protein expression is detected using a Western blot of cells lysates probed
with antibodies to the
TRB protein. In another embodiment, reverse transcriptase polymerase chain
reactions (RT-
PCR) are used to confirm the presence of the inactivating genetic
modification.
1004071 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise knock out of TRB expression, such that the cells are TRB-. In some
embodiments, the
engineered and/or hypoimmunogenic cells outlined herein introduce an indel
into the TRB gene
locus, such that the cells are TRB'"""'. In some embodiments, the engineered
and/or
hypoimmunogenic cells outlined herein comprise knock down of TRB expression,
such that the
cells are TRBknock down
G. CD142
[00408] In certain embodiments, the technologies disclosed herein modulate
(e.g., reduce or
eliminate) the expression of CD142, which is also known as tissue factor,
factor III, and F3. In
some embodiments, the modulation occurs using a gene editing system (e.g.
CRISPR/Cas
system).
[00409] In some embodiments, the target polynucleotide sequence is CD142 or a
variant of
CD142. In some embodiments, the target polynucleotide sequence is a homolog of
CD142. In
some embodiments, the target polynucleotide sequence is an ortholog of CD142.
[00410] In some embodiments, the cells outlined herein comprise a genetic
modification
targeting the CD142 gene. In some embodiments, the genetic modification
targeting the CD142
gene by the rare-cutting endonuclease comprises a Cas protein or a
polynucleotide encoding a
Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for
specifically targeting
the CD142 gene. Useful methods for identifying gRNA sequences to target CD142
are
described below.
86
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1004111 Assays to test whether the CD142 gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the CD142
gene by PCR and
the reduction of CD142 expression can be assays by FACS analysis. In another
embodiment,
CD142 protein expression is detected using a Western blot of cells lysates
probed with
antibodies to the CD142 protein. In another embodiment, reverse transcriptase
polymerase chain
reactions (RT-PCR) are used to confirm the presence of the inactivating
genetic modification.
1004121 Useful genomic, polynucleotide and polypeptide information about the
human CD142
are provided in, for example, the GeneCard Identifier GC01M094530, HGNC No.
3541, NCBI
Gene ID 2152, NCBI RefSeq Nos. NM 001178096.1, NM 001993.4, NP 001171567.1,
and
NP 001984.1, UniProt No. P13726, and the like.
H. RHD
1004131 In some embodiments, the technologies disclosed herein modulate (e.g.,
reduce or
eliminate) the expression of RhD antigen by targeting and modulating (e.g.,
reducing or
eliminating) expression of the RHD gene. In some embodiments, the modulation
occurs using a
gene editing system (e.g. CRISPR/Cas system). In some embodiments, the cell
has a reduced
ability to induce an innate and/or an adaptive immune response in a recipient
subject.
1004141 In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of REED gene. In some embodiments, the target polynucleotide sequence
is a homolog of
RHD gene. In some embodiments, the target polynucleotide sequence is an
ortholog of RHD
gene.
1004151 In some embodiments, the cells described herein comprise gene
modifications at the
gene locus encoding the RhD antigen protein. In other words, the cells
comprise a genetic
modification at the RHD locus. In some instances, the nucleotide sequence
encoding the RhD
antigen protein is set forth in RefSeq. Nos. NM 001127691.2, NM 001282868.1,
NM 001282869.1, NM 001282871.1, or NM 016124.4, or in Genbank No. L08429. in
some
instances, the RHD gene locus is described in NCBI Gene ID No.6007. In certain
cases, the
amino acid sequence of RhD antigen protein is depicted as NCBI GenBank No.
AAA02679.1.
Additional descriptions of the RhD protein and gene locus can be found in
Uniprot No. Q02161,
HGNC Ref No. 10009, and OMIM Ref. No. 111680.
87
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1004161 In some embodiments, the cells outlined herein comprise a genetic
modification
targeting the REID gene. In some embodiments, the genetic modification
targeting the RI-ID gene
is generated by gene editing the RHD gene using gene editing tools such as but
not limited to
CRISPR/Cas, TALE- nucleases, zinc finger nucleases, other viral based gene
editing system, or
RNA interference. In some embodiments, the gene editing targets the coding
sequence of the
RHD gene. In some instances, the cells do not generate a functional RHD gene
product. In the
absence of the RHD gene product, the cells completely lack an Rh blood group
antigen.
1004171 In some embodiments, the genetic modification targeting the RHD gene
by the rare-
cutting endonuclease comprises a Cas protein or a polynucleotide encoding a
Cas protein, and at
least one guide ribonucleic acid (gRNA) sequence for specifically targeting
the RHD gene.
Useful methods for identifying gRNA sequences to target RHD are described
below.
1004181 Assays to test whether the RHD gene has been inactivated are known and
described
herein. In some embodiments, the resulting genetic modification of the RHD
gene by PCR and
the reduction of RHD expression can be assays by FACS analysis. In another
embodiment, RhD
protein expression is detected using a Western blot of cells lysates probed
with antibodies to the
RhD protein. In another embodiment, reverse transcriptasc polymerase chain
reactions (RT-
PCR) are used to confirm the presence of the inactivating genetic
modification.
I. CTLA-4
1004191 In some embodiments, the target polynucleotide sequence is CTLA-4 or a
variant of
CTLA-4. In some embodiments, the target polynucleotide sequence is a homolog
of CTLA-4.
In some embodiments, the target polynucleotide sequence is an ortholog of CTLA-
4.
1004201 In some embodiments, the cells outlined herein comprise a genetic
modification
targeting the CTLA-4 gene. In certain embodiments, primary T cells comprise a
genetic
modification targeting the CTLA-4 gene. The genetic modification can reduce
expression of
CTLA-4 polynucleotides and CTLA-4 polypeptides in T cells includes primary T
cells and
CAR-T cells. In some embodiments, the genetic modification targeting the CTLA-
4 gene by the
rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding
a Cas protein,
and at least one guide ribonucleic acid (gRNA) sequence for specifically
targeting the CTLA-4
gene. Useful methods for identifying gRNA sequences to target CTLA-4 are
described below.
88
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1004211 Assays to test whether the CTLA-4 gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the CTLA-4
gene by PCR
and the reduction of CTLA-4 expression can be assays by FACS analysis. In
another
embodiment, CTLA-4 protein expression is detected using a Western blot of
cells lysates probed
with antibodies to the CTLA-4 protein. In another embodiment, reverse
transcriptase
polymerase chain reactions (RT-PCR) are used to confirm the presence of the
inactivating
genetic modification.
1004221 Useful genomic, polynucleotide and polypeptide information about the
human CTLA-4
are provided in, for example, the GeneCard Identifier GCO2P203867, HGNC No.
2505, NCBI
Gene ID 1493, NCBI RefSeq Nos. NM 005214.4, NM 001037631.2, NP 001032720.1 and

NP 005205.2, UniProt No. P16410, and the like.
J. PD-1
1004231 In some embodiments, the target polynucleotide sequence is PD-1 or a
variant of PD-
1. In some embodiments, the target polynucleotide sequence is a homolog of PD-
1. In some
embodiments, the target polynucleotide sequence is an ortholog of PD-1.
1004241 In some embodiments, the cells outlined herein comprise a genetic
modification
targeting the gene encoding the programmed cell death protein 1 (PD- I)
protein or the PDCD1
gene. In certain embodiments, primary T cells comprise a genetic modification
targeting the
PDCD1 gene. The genetic modification can reduce expression of PD-1
polynucleotides and PD-
1 polypeptides in T cells includes primary T cells and CAR-T cells. In some
embodiments, the
genetic modification targeting the PDCD1 gene by the rare-cutting endonuclease
comprises a
Cas protein or a polynucleotide encoding a Cos protein, and at least one guide
ribonucleic acid
(gRNA) sequence for specifically targeting the PDCD1 gene. Useful methods for
identifying
gRNA sequences to target PD-1 are described below.
1004251 Assays to test whether the PDCD1 gene has been inactivated are known
and described
herein. In some embodiments, the resulting genetic modification of the PDCD1
gene by PCR
and the reduction of PD-1 expression can be assays by FACS analysis. In
another embodiment,
PD-1 protein expression is detected using a Western blot of cells lysates
probed with antibodies
to the PD-1 protein. In another embodiment, reverse transcriptase polymerase
chain reactions
(RT-PCR) are used to confirm the presence of the inactivating genetic
modification.
89
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1004261 Useful genomic, polynucleotide and polypeptide information about human
PD-1
including the PDCD1 gene are provided in, for example, the GeneCard Identifier

GCO2M241849, HGNC No. 8760, NCBI Gene ID 5133, Uniprot No. Q15116, and NCBI
RefSeq
Nos. NM 005018.2 and NP 005009.2.
K. CD47
1004271 In some embodiments, the present disclosure provides a cell or
population thereof that
has been modified to express the tolerogenic factor (e.g., immunomodulatory
polypeptide)
CD47. In some embodiments, the present disclosure provides a method for
altering a cell
genome to express CD47. In some embodiments, the stem cell expresses exogenous
CD47. In
some instances, the cell expresses an expression vector comprising a
nucleotide sequence
encoding a human CD47 polypeptide. In some embodiments, the cell is
genetically modified to
comprise an integrated exogenous polynucleotide encoding CD47 using homology-
directed
repair. In some instances, the cell expresses a nucleotide sequence encoding a
human CD47
polypeptide such that the nucleotide sequence is inserted into at least one
allele of a safe harbor
or target locus. In some instances, the cell expresses a nucleotide sequence
encoding a human
CD47 polypeptide such that the nucleotide sequence is inserted into at least
one allele of an
AAVS 1 locus. In some instances, the cell expresses a nucleotide sequence
encoding a human
CD47 polypeptide such that the nucleotide sequence is inserted into at least
one allele of a safe
harbor or target locus. In some instances, the cell expresses a nucleotide
sequence encoding a
human CD47 polypeptide such that the nucleotide sequence is inserted into at
least one allele of
an CCR5 locus. In some instances, the cell expresses a nucleotide sequence
encoding a human
CD47 polypeptide wherein the nucleotide sequence is inserted into at least one
allele of an
AAVS1 locus. In some instances, the cell expresses a nucleotide sequence
encoding a human
CD47 polypeptide wherein the nucleotide sequence is inserted into at least one
allele of an CCR5
locus. In some instances, the cell expresses a nucleotide sequence encoding a
human CD47
polypeptide wherein the nucleotide sequence is inserted into at least one
allele of a safe harbor or
target locus, such as, but not limited to, a CCR5 gene locus, a CXCR4 gene
locus, a PPP1R12C
gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene locus, a
Rosa gene
locus, an F3 (CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1
(CD91) gene
locus, a HMGB1 gene locus, an ABO gene locus, a R_HD gene locus, a FUT1 locus,
and a
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
KDM5D gene locus. In some instances, the cell expresses a nucleotide sequence
encoding a
human CD47 polypeptide wherein the nucleotide sequence is inserted into at
least one allele of a
TRAC locus.
1004281 CD47 is a leukocyte surface antigen and has a role in cell adhesion
and modulation of
integrins. It is expressed on the surface of a cell and signals to circulating
macrophages not to
eat the cell.
1004291 In some embodiments, the cell outlined herein comprises a nucleotide
sequence
encoding a CD47 polypeptide has at least 95% sequence identity (e.g., 95%,
96%, 97%, 98%,
99%, or more) to an amino acid sequence as set forth in NCBI Ref. Sequence
Nos. NP 001768.1
and NP 942088.1. In some embodiments, the cell outlined herein comprises a
nucleotide
sequence encoding a CD47 polypeptide having an amino acid sequence as set
forth in NCBI Ref.
Sequence Nos. NP 001768.1 and NP 942088.1. In some embodiments, the cell
comprises a
nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%,
86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the
sequence
set forth in NCBI Ref Nos. NIVI 001777.3 and NM 198793.2. In some embodiments,
the cell
comprises a nucleotide sequence for CD47 as set forth in NCBI Ref. Sequence
Nos.
NM 001777.3 and NM 198793.2. In some embodiments, the nucleotide sequence
encoding a
CD47 polynucleotide is a codon optimized sequence. In some embodiments, the
nucleotide
sequence encoding a CD47 polynucleotide is a human codon optimized sequence.
1004301 In some embodiments, the cell comprises a CD47 polypeptide having at
least 95%
sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to an amino acid
sequence as set
forth in NCBI Ref Sequence Nos. NP 001768.1 and NP 942088.1. In some
embodiments, the
cell outlined herein comprises a CD47 polypeptide having an amino acid
sequence as set forth in
NCB' Ref Sequence Nos. NP 001768.1 and NP 942088.1.
1004311 Exemplary amino acid sequences of human CD47 with a signal sequence
and without a
signal sequence are provided in Table 1.
Table 1. Amino acid sequences of human C047
Protein SEQ Sequence
Amino
ID acid
NO:
residues
Human 13 QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNT FEN aa 19-323
CD47 YVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVS
91
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
(without QLLKGDASLKMDKSDAVSHTGNYTCEVTELTREG
signal ETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGI
sequence) KTLKYRSGGMDEKTIALLYAGLVITVIVIVGAILFVP
GEYSLKNATGLGLIVTSTGILILLHYYVF STAIGLTSF
VIAILVIQVIAYILAVVGLSLCIAACIPMEIGPLLISGL
SILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLN
AFKESKGM_MNDE
Human 14 MWPLVAALLLGSACCGSAQLLENKTKSVEFTFCND aa 1-323
CD47 (with TVVIPCFV'TNMEAQNTTEVYVKWKFKGRDIYTEDG
signal ALNKSTYPTDESSAKIEVSQLLKGDASLKMDKSDA
sequence) VSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNE
NILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIAL
LVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTS
TGILILLHYYVF STAIGLTSEVIAILVIQVIAY1LAVVG
LSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKEV
ASNQKTIQPPRKAVEEPLNAFKESKGMIVINDE
[00432] In some embodiments, the cell comprises a CD47 polypeptide having at
least 95%
sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid
sequence of SEQ
ID NO:13. In some embodiments, the cell comprises a CD47 polypeptide having
the amino acid
sequence of SEQ ID NO:13. In some embodiments, the cell comprises a CD47
polypeptide
having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more)
to the amino
acid sequence of SEQ ID NO:14. In some embodiments, the cell comprises a CD47
polypeptide
having the amino acid sequence of SEQ ID NO:14.
[00433] In some embodiments, the cell comprises a nucleotide sequence encoding
a CD47
polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%,
99%, or more) to
the amino acid sequence of SEQ ID NO:13. In some embodiments, the cell
comprises a
nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence
of SEQ ID
NO:13. In some embodiments, the cell comprises a nucleotide sequence encoding
a CD47
polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%,
99%, or more) to
the amino acid sequence of SEQ ID NO:14. In some embodiments, the cell
comprises a
nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence
of SEQ ID
NO: 14. In some embodiments, the nucleotide sequence is codon optimized for
expression in a
particular cell.
[00434] In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas
system or any
of the gene editing systems described herein) is used to facilitate the
insertion of a
92
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
polynucleotide encoding CD47, into a genomic locus of the hypoimmunogenic
cell. In some
cases, the polynucleotide encoding CD47 is inserted into a safe harbor or
target locus, such as
but not limited to, an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA,
MICB,
LRP1 (CD91), ABO, RHD, FUT1, or KDM5D gene locus. In some
embodiments, the
polynucleotide encoding CD47 is inserted into a B2M gene locus, a CIITA gene
locus, a TRAC
gene locus, or a TRB gene locus. In some embodiments, the polynucleotide
encoding CD47 is
inserted into any one of the gene loci depicted in Table 21 provided herein.
In certain
embodiments, the polynucleotide encoding CD47 is operably linked to a
promoter.
1004351 In another embodiment, CD47 protein expression is detected using a
Western blot of
cell lysates probed with antibodies against the CD47 protein. In another
embodiment, reverse
transcriptase polymerase chain reactions (RT-PCR) are used to confirm the
presence of the
exogenous CD47 mRNA.
L. CD24
1004361 In some embodiments, the present disclosure provides a cell or
population thereof that
has been modified to express the tolerogenic factor (e.g., immunomodulatory
polypeptide)
CD24. In some embodiments, the present disclosure provides a method for
altering a cell
genome to express CD24. In some embodiments, the stem cell expresses exogenous
CD24. In
some instances, the cell expresses an expression vector comprising a
nucleotide sequence
encoding a human CD24 polypeptide.
1004371 CD24 which is also referred to as a heat stable antigen or small-cell
lung cancer cluster
4 antigen is a glycosylated glycosylphosphatidylinositol-anchored surface
protein (Pirruccello et
al., J Immunol, 1986, 136, 3779-3784, Chen et al., Glycobiology, 2017, 57, 800-
806). It binds to
Siglec-10 on innate immune cells. Recently it has been shown that CD24 via
Siglec-10 acts as
an innate immune checkpoint (Barkal et al., Nature, 2019, 572, 392-396).
1004381 In some embodiments, the cell outlined herein comprises a nucleotide
sequence
encoding a CD24 polypeptide has at least 95% sequence identity (e.g., 95%,
96%, 97%, 98%,
99%, or more) to an amino acid sequence set forth in NCBI Ref Nos. NP
001278666.1,
NP 001278667.1, NP 001278668.1, and NP 037362.1. In some embodiments, the cell
outlined
herein comprises a nucleotide sequence encoding a CD24 polypeptide having an
amino acid
93
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
sequence set forth in NCBI Ref. Nos. NP 001278666.1, NP 001278667.1, NP
001278668.1,
and NP 037362.1.
1004391 In some embodiments, the cell comprises a nucleotide sequence having
at least 85%
sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%,
97%, 98%, 99%, or more) to the sequence set forth in NCBI Ref. Nos. NM
00129737.1,
NM 00129738.1, NM 001291739.1, and NM 013230.3. In some embodiments, the cell
comprises a nucleotide sequence as set forth in NCBI Ref. Nos. NM 00129737.1,
NM 00129738.1, NM 001291739.1, and NM 013230.3.
1004401 In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas
system or any
of the gene editing systems described herein) is used to facilitate the
insertion of a
polynucleotide encoding CD24, into a genomic locus of the hypoimmunogenic
cell. In some
cases, the polynucleotide encoding CD24 is inserted into a safe harbor or
target locus, such as
but not limited to, an AAVS I, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA,
MICB,
LRP1 (CD91), 111VIGB1, ABO, RED, FUT1, or KDM5D gene locus. In some
embodiments, the
polynucleotide encoding CD24 is inserted into a B2M gene locus, a CIITA gene
locus, a TRAC
gene locus, or a TRB gene locus. In some embodiments, the polynucleotide
encoding CD24 is
inserted into any one of the gene loci depicted in Table 20 provided herein.
In certain
embodiments, the polynucleotide encoding CD24 is operably linked to a
promoter.
1004411 In another embodiment, CD24 protein expression is detected using a
Western blot of
cells lysates probed with antibodies against the CD24 protein. In another
embodiment, reverse
transcriptase polymerase chain reactions (RT-PCR) are used to confirm the
presence of the
exogenous CD24 mRNA.
1004421 In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas
system or any
of the gene editing systems described herein) is used to facilitate the
insertion of a
polynucleotide encoding CD24, into a genomic locus of the hypoimmunogenic
cell. In some
cases, the polynucleotide encoding CD24 is inserted into a safe harbor or
target locus, such as
but not limited to, an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as
CD142),
MICA, MICB, LRP I (also known as CD91), EIMGB I, ABO, REID, FUT I, or KDM5D
gene
locus. In some embodiments, the polynucleotide encoding CD24 is inserted into
a B2M gene
locus, a CIITA gene locus, a TRAC gene locus, or a TRB gene locus. In some
embodiments, the
polynucleotide encoding CD24 is inserted into any one of the gene loci
depicted in Table 20
94
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
provided herein. In certain embodiments, the polynucleotide encoding CD24 is
operably linked
to a promoter.
M. DUX4
1004431 In some embodiments, the present disclosure provides a cell (e.g.,
stem cell, induced
pluripotent stem cell, differentiated cell, hematopoietic stem cell, primary T
cell or CAR-T cell)
or population thereof comprising a genome modified to increase expression of a
tolerogenic or
immunosuppressive factor such as DUX4. In some embodiments, the present
disclosure
provides a method for altering a cell's genome to provide increased expression
of DUX4. In
some embodiments, the disclosure provides a cell or population thereof
comprising exogenously
expressed DUX4 proteins. In some embodiments, increased expression of DUX4
suppresses,
reduces or eliminates expression of one or more of the following MHC I
molecules: HLA-A,
HLA-B, and HLA-C.
1004441 DUX4 is a transcription factor that is active in embryonic tissues and
induced
pluripotent stem cells, and is silent in normal, healthy somatic tissues (Feng
et al., 2015, ELife4;
De Taco et al., 2017, Nat Genet, 49, 941-945; Hendrickson et al., 2017, Nat
Genet, 49, 925-934,
Snider et al., 2010, PLoS Genet, e1001181; Whiddon et al., 2017, Nat Genet).
DUX4 expression
acts to block IFN-gamma mediated induction of MHC class I gene expression
(e.g., expression
of B2111, HLA-A, HLA-B, and HLA-C). DUX4 expression has been implicated in
suppressed
antigen presentation by MEC class I (Chew et al., Developmental Cell, 2019,
50, 1-14). DUX4
functions as a transcription factor in the cleavage-stage gene expression
(transcriptional)
program. Its target genes include, but are not limited to, coding genes,
noncoding genes, and
repetitive elements.
1004451 There are at least two isoforms of DUX4, with the longest isoform
comprising the
DUX4 C-terminal transcription activation domain. The isoforms are produced by
alternative
splicing. See, e.g., Geng et al., 2012, Dev Cell, 22, 38-51; Snider et al.,
2010, PLoS Genet,
e1001181. Active isoforms for DUX4 comprise its N-terminal DNA-binding domains
and its C-
terminal activation domain. See, e.g., Choi et al., 2016, Nucleic Acid Res,
44, 5161-5173.
1004461 It has been shown that reducing the number of CpG motifs of DUX4
decreases
silencing of a DUX4 transgene (Jagannathan et al., Human Molecular Genetics,
2016,
25(20):4419-4431). The nucleic acid sequence provided in Jagannathan et al.,
supra represents a
codon altered sequence of DUX4 comprising one or more base substitutions to
reduce the total
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
number of CpG sites while preserving the DUX4 protein sequence. The nucleic
acid sequence is
commercially available from Addgene, Catalog No. 99281.
[00447] In many embodiments, at least one or more polynucleotides may be
utilized to
facilitate the exogenous expression of DUX4 by a cell, e.g., a stem cell,
induced pluripotent stem
cell, differentiated cell, hematopoietic stem cell, primary T cell or CAR-T
cell.
[00448] In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas
system or any
of the gene editing systems described herein) is used to facilitate the
insertion of a
polynucleotide encoding DUX4, into a genomic locus of the hypoimmunogenic
cell. In some
cases, the polynucleotide encoding DUX4 is inserted into a safe harbor or
target locus, such as
but not limited to, an AAVS I, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA,
MICB,
LRP1 (CD91), 111VIGBI, ABO, REID, FUT I, or KDM5D gene locus. In some
embodiments, the
polynucleotide encoding DUX4 is inserted into a B2M gene locus, a CIITA gene
locus, a TRAC
gene locus, or a TRB gene locus. In some embodiments, the polynucleotide
encoding DUX4 is
inserted into any one of the gene loci depicted in Table 20 provided herein.
In certain
embodiments, the polynucleotide encoding DUX4 is operably linked to a
promoter.
[00449] In some embodiments, the polynucleotide sequence encoding DUX4
comprises a
polynucleotide sequence comprising a codon altered nucleotide sequence of DUX4
comprising
one or more base substitutions to reduce the total number of CpG sites while
preserving the
DUX4 protein sequence. In some embodiments, the polynucleotide sequence
encoding DUX4
comprising one or more base substitutions to reduce the total number of CpG
sites has at least
85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%
or 100%) sequence identity to SEQ ID NO:1 of PCT/US2020/44635, filed July 31,
2020. In
some embodiments, the polynucleotide sequence encoding DUX4 is SEQ ID NO.1 of
PCT/US2020/44635.
1004501 In some embodiments, the polynucleotide sequence encoding DUX4 is a
nucleotide
sequence encoding a polypeptide sequence haying at least 95% (e.g., 95%, 96%,
97%, 98%, 99%
or 100%) sequence identity to a sequence selected from a group including SEQ
ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ
ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,
SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID
NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID
96
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, as provided in
PCT/US2020/44635. In some embodiments, the polynucleotide sequence encoding
DUX4 is a
nucleotide sequence encoding a polypeptide sequence is selected from a group
including SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
SEQ
ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID
NO.25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. Amino acid
sequences set
forth as SEQ ID NOS:2-29 are shown in Figure 1A-1G of PCT/U52020/44635.
1004511 In some instances, the DUX4 polypeptide comprises an amino acid
sequence having at
least 95% sequence identity to the sequence set forth in GenBank Accession No.
ACN62209.1 or
an amino acid sequence set forth in GenBank Accession No. ACN62209.1. In some
instances,
the DUX4 polypeptide comprises an amino acid sequence having at least 95%
sequence identity
to the sequence set forth in NCBI RefSeq No. NP 001280727.1 or an amino acid
sequence set
forth in NCBI RefSeq No. NP 001280727.1. In some instances, the DUX4
polypeptide
comprises an amino acid sequence having at least 95% sequence identity to the
sequence set
forth in GenBank Accession No. ACP30489.1 or an amino acid sequence set forth
in GenBank
Accession No. ACP30489.1. In some instances, the DUX4 polypeptide comprises an
amino acid
sequence having at least 95% sequence identity to the sequence set forth in
UniProt No.
POCJ85.1 or an amino acid sequence set forth in UniProt No. POCJ85.1. In some
instances, the
DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence
identity to
the sequence set forth in GenBank Accession No. AUA60622.1 or an amino acid
sequence set
forth in GenBank Accession No. AUA60622.1. In some instances, the DUX4
polypeptide
comprises an amino acid sequence having at least 95% sequence identity to the
sequence set
forth in GenBank Accession No. ADK24683.1 or an amino acid sequence set forth
in GenBank
Accession No. ADK24683.1. In some instances, the DUX4 polypeptide comprises an
amino
acid sequence having at least 95% sequence identity to the sequence set forth
in GenBank
Accession No. ACN62210.1 or an amino acid sequence set forth in GenBank
Accession No.
ACN62210.1. In some instances, the DUX4 polypeptide comprises an amino acid
sequence
having at least 95% sequence identity to the sequence set forth in GenBank
Accession No.
ADK24706.1 or an amino acid sequence set forth in GenBank Accession No.
ADK24706.1. In
97
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some instances, the DUX4 polypeptide comprises an amino acid sequence haying
at least 95%
sequence identity to the sequence set forth in GenBank Accession No.
ADK24685.1 or an amino
acid sequence set forth in GenBank Accession No. ADK24685.1. In some
instances, the DUX4
polypeptide comprises an amino acid sequence haying at least 95% sequence
identity to the
sequence set forth in GenBank Accession No. ACP30488.1 or an amino acid
sequence set forth
in GenBank Accession No. ACP30488.1. In some instances, the DUX4 polypeptide
comprises
an amino acid sequence haying at least 95% sequence identity to the sequence
set forth in
GenBank Accession No. ADK24687.1 or an amino acid sequence set forth in
GenBank
Accession No. ADK24687.1. In some instances, the DUX4 polypeptide comprises an
amino acid
sequence haying at least 95% sequence identity to the sequence set forth in
GenBank Accession
No. ACP30487.1 or an amino acid sequence set forth in GenBank Accession No.
ACP30487.1.
In some instances, the DUX4 polypeptide comprises an amino acid sequence
haying at least 95%
sequence identity to the sequence set forth in GenBank Accession No.
ADK24717.1 or an amino
acid sequence set forth in GenBank Accession No. ADK24717.1. In some
instances, the DUX4
polypeptide comprises an amino acid sequence haying at least 95% sequence
identity to the
sequence set forth in GenBank Accession No. ADK24690.1 or an amino acid
sequence set forth
in GenBank Accession No. ADK24690.1. In some instances, the DUX4 polypeptide
comprises
an amino acid sequence haying at least 95% sequence identity to the sequence
set forth in
GenBank Accession No. ADK24689.1 or an amino acid sequence set forth in
GenBank
Accession No. ADK24689.1. In some instances, the DUX4 polypeptide comprises an
amino acid
sequence haying at least 95% sequence identity to the sequence set forth in
GenBank Accession
No. ADK24692.1 or an amino acid sequence set forth in GenBank Accession No.
ADK24692.1.
In some instances, the DUX4 polypeptide comprises an amino acid sequence
haying at least 95%
sequence identity to the sequence set forth in GenBank Accession No.
A_DK24693.1 or an amino
acid sequence of set forth in GenBank Accession No. ADK24693.1. In some
instances, the
DUX4 polypeptide comprises an amino acid sequence haying at least 95% sequence
identity to
the sequence set forth in GenBank Accession No. ADK24712.1 or an amino acid
sequence set
forth in GenBank Accession No. ADK24712.1. In some instances, the DUX4
polypeptide
comprises an amino acid sequence haying at least 95% sequence identity to the
sequence set
forth in GenBank Accession No. ADK24691.1 or an amino acid sequence set forth
in GenBank
Accession No. ADK24691.1. In some instances, the DUX4 polypeptide comprises an
amino acid
98
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
sequence having at least 95% sequence identity to the sequence set forth in
UniProt No.
POCJ87.1 or an amino acid sequence of set forth in UniProt No. POCJ87.1. In
some instances, the
DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence
identity to
the sequence set forth in GenBank Accession No. ADK24714.1 or an amino acid
sequence set
forth in GenBank Accession No. ADK24714.1. In some instances, the DUX4
polypeptide
comprises an amino acid sequence having at least 95% sequence identity to the
sequence set
forth in GenBank Accession No. ADK24684.1 or an amino acid sequence of set
forth in
GenBank Accession No. ADK24684.1. In some instances, the DUX4 polypeptide
comprises an
amino acid sequence having at least 95% sequence identity to the sequence set
forth in GenBank
Accession No. ADK24695.1 or an amino acid sequence set forth in GenBank
Accession No.
ADK24695.1. In some instances, the DUX4 polypeptide comprises an amino acid
sequence
having at least 95% sequence identity to the sequence set forth in GenBank
Accession No.
ADK24699.1 or an amino acid sequence set forth in GenBank Accession No.
ADK24699.1. In
some instances, the DUX4 polypeptide comprises an amino acid sequence having
at least 95%
sequence identity to the sequence set forth in NCBI RefSeq No. NP 001768.1 or
an amino acid
sequence set forth in NCBI RefSeq No. NP 001768. In some instances, the DUX4
polypeptide
comprises an amino acid sequence having at least 95% sequence identity to the
sequence set
forth in NCBI RefSeq No. NP 942088.1 or an amino acid sequence set forth in
NCBI RefSeq
No. NP 942088.1. In some instances, the DUX4 polypeptide comprises an amino
acid sequence
having at least 95% sequence identity to SEQ ID NO:28 provided in
PCT/US2020/44635 or an
amino acid sequence of SEQ ID NO:28 provided in PCT/U52020/44635. In some
instances, the
DUX4 polypeptide comprises an amino acid sequence having at least 95% sequence
identity to
SEQ ID NO:29 provided in PCT/U52020/44635 or an amino acid sequence of SEQ ID
NO:29
provided in PCT/US2020/44635.
1004521 In other embodiments, expression of tolerogenic factors is facilitated
using an
expression vector. In some embodiments, the expression vector comprises a
polynucleotide
sequence encoding DUX4 is a codon altered sequence comprising one or more base
substitutions
to reduce the total number of CpG sites while preserving the DUX4 protein
sequence. In some
cases, the codon altered sequence of DUX4 comprises SEQ ID NO:1 of
PCT/US2020/44635. In
some cases, the codon altered sequence of DUX4 is SEQ ID NO:1 of
PCT/US2020/44635. In
other embodiments, the expression vector comprises a polynucleotide sequence
encoding DUX4
99
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
comprising SEQ ID NO:1 of PCT/US2020/44635. In some embodiments, the
expression vector
comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence
having at least
95% sequence identity to a sequence selected from a group including SEQ ID
NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,
SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20,
SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID
NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT/US2020/44635. In
some
embodiments, the expression vector comprises a polynucleotide sequence
encoding a DUX4
polypeptide sequence selected from a group including SEQ ID NO:2, SEQ ID NO:3,
SEQ ID
NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,
SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, and SEQ ID NO:29 of PCT/US2020/44635.
1004531 An increase of DUX4 expression can be assayed using known techniques,
such as
Western blots, ELISA assays, FACS assays, immunoassays, and the like.
N. Additional Tolerogenic Factors
1004541 In certain embodiments, one or more tolerogenic factors can be
inserted or reinserted
into genome-edited cells to create immune-privileged universal donor cells,
such as universal
donor stem cells, universal donor T cells, or universal donor cells. In
certain embodiments, the
engineered and/or hypoimmunogenic cells disclosed herein have been further
modified to
express one or more tolerogenic factors. Exemplary tolerogenic factors
include, without
limitation, one or more of CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59,
CD200,
HLA-C, HLA-E, fiLA-E heavy chain, HLA-G, PD-Li, Ill01, CILA4-1g, C1-Inhibitor,
IL-10,
IL-35, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fe receptor, IL15-
RF, and
Serpinb9. In some embodiments, the tolerogenic factors are selected from the
group consisting
of CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, 1D01, CTLA4-Ig, IL-
10, IL-
35, FasL, Serpinb9, CCL21, CCL22, and Mfge8. In some embodiments, the
tolerogenic factors
are selected from the group consisting of DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-
L1,
CTLA-4-Ig, Cl-inhibitor, and IL-35. In some embodiments, the tolerogenic
factors are selected
100
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
from the group consisting of HLA-C, HLA-E, HLA-F, HLA-G, PD-Li, CTLA-4-Ig, CI-
inhibitor, and IL-35. In some embodiments, the tolerogenic factors are
selected from a group
including CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E,

HLA-E heavy chain, HLA-G, PD-L1, IDOL CTLA4-Ig, Cl-Inhibitor, IL-10, IL-35,
FasL,
CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and
Serpinb9.
[00455] Useful genomic, polynucleotide and polypeptide information about human
CD27
(which is also known as CD27L receptor, Tumor Necrosis Factor Receptor
Superfamily Member
7, TNFSF7, T Cell Activation Antigen S152, Tp55, and T14) are provided in, for
example, the
GeneCard Identifier GC12P008144, HGNC No. 11922, NCBI Gene ID 939, Uniprot No.

P26842, and NCBI RefSeq Nos. NM 001242.4 and NP 001233.1.
1004561 Useful genomic, polynucleotide and polypeptide information about human
CD46 are
provided in, for example, the GeneCard Identifier GC01P207752, HGNC No. 6953,
NCBI Gene
ID 4179, Uniprot No. P15529, and NCBI RefSeq Nos. NM 002389.4, NM 153826.3,
NM 172350.2, NM 172351.2, NM 172352.2 NP 758860.1, NM 172353.2, NM 172359.2,
NM 172361.2, NP 002380.3, NP 722548.1, NP 758860.1, NP 758861.1, NP 758862.1,
NP 758863.1, NP 758869.1, and NP 758871.1.
[00457] Useful genomic, polynucleotide and polypeptide information about human
CD55 (also
known as complement decay-accelerating factor) are provided in, for example,
the GeneCard
Identifier GC01P207321, HGNC No. 2665, NCBI Gene ID 1604, Uniprot No. P08174,
and
NCBI RefSeq Nos. NM 000574.4, NM 001114752.2, NM 001300903.1, NM 001300904.1,
NP 000565.1, NP 001108224.1, NP 001287832.1, and NP 001287833.1.
[00458] Useful genomic, polynucleotide and polypeptide information about human
CD59 are
provided in, for example, the GeneCard Identifier GC11M033704, HGNC No. 1689,
NCBI Gene
ID 966, Uniprot No. P13987, and NCBI RefSeq Nos. NP 000602.1, NM 000611.5,
NP 001120695.1, NM 001127223.1, NP 001120697.1, NM 001127225.1, NP
001120698.1,
NM 001127226.1, NP 001120699.1, NM 001127227.1, NP 976074.1, NM 203329.2,
NP 976075.1, NM 203330.2, NP 976076.1, and NM 203331.2.
[00459] Useful genomic, polynucleotide and polypeptide information about human
CD200 are
provided in, for example, the GeneCard Identifier GC03P112332, HGNC No. 7203,
NCBI Gene
ID 4345, Uniprot No. P41217, and NCBI RefSeq Nos. NP 001004196.2, NM
001004196.3,
101
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NP 001305757.1, NM 001318828.1, NP 005935.4, NM 005944.6, XP 005247539.1, and
XM 005247482.2.
[00460] Useful genomic, polynucleotide and polypeptide information about human
HLA-C are
provided in, for example, the GeneCard Identifier GC06M031272, HGNC No. 4933,
NCBI Gene
ID 3107, Uniprot No. P10321, and NCBI RefSeq Nos. NP 002108.4 and NM 002117.5.

[00461] Useful genomic, polynucleotide and polypeptide information about human
HLA-E are
provided in, for example, the GeneCard Identifier GCO6P047281, HGNC No. 4962,
NCBI Gene
ID 3133, Uniprot No. P13747, and NCBI RefSeq Nos. NP 005507.3 and NM 005516.5.

[00462] Useful genomic, polynucleotide and polypeptide information about human
HLA-G are
provided in, for example, the GeneCard Identifier GC06P047256, HGNC No. 4964,
NCBI Gene
ID 3135, Uniprot No. P17693, and NCBI RefSeq Nos. NP 002118.1 and NM 002127.5.

[00463] Useful genomic, polynucleotide and polypeptide information about human
PD-Li or
CD274 are provided in, for example, the GeneCard Identifier GC09P005450, HGNC
No. 17635,
NCBI Gene ID 29126, Uniprot No. Q9NZQ7, and NCBI RefSeq Nos. NP 001254635.1,
NM 001267706.1, NP 054862.1, and NM 014143.3.
[00464] Useful genomic, polynucleotide and polypeptide information about human
IDO1 are
provided in, for example, the GeneCard Identifier GC08P039891, HGNC No. 6059,
NCBI Gene
ID 3620, Uniprot No. P14902, and NCBI RefSeq Nos. NP 002155.1 and NM 002164.5.

[00465] Useful genomic, polynucleotide and polypeptide information about human
IL-10 are
provided in, for example, the GeneCard Identifier GC01M206767, HGNC No. 5962,
NCBI Gene
ID 3586, Uniprot No. P22301, and NCBI RefSeq Nos. NP 000563.1 and NM 000572.2.

[00466] Useful genomic, polynucleotide and polypeptide information about human
Fas ligand
(which is known as FasL, FASLG, CD178, TNFSF6, and the like) are provided in,
for example,
the GeneCard Identifier GC01P172628, HGNC No. 11936, NCBI Gene ID 356, Uniprot
No.
P48023, and NCBI RefSeq Nos. NP 000630.1, NM 000639.2, NP 001289675.1, and
NM 001302746.1.
[00467] Useful genomic, polynucleotide and polypeptide information about human
CCL21 are
provided in, for example, the GeneCard Identifier GC09M034709, HGNC No. 10620,
NCBI
Gene ID 6366, Uniprot No. 000585, and NCBI RefSeq Nos. NP 002980.1 and NM
002989.3.
[00468] Useful genomic, polynucleotide and polypeptide information about human
CCL22 are
provided in, for example, the GeneCard Identifier GC16P057359, HGNC No. 10621,
NCBI
102
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
Gene ID 6367, Uniprot No. 000626, and NCBI RefSeq Nos. NP 002981.2, NM
002990.4,
XP 016879020.1, and XM 017023531.1.
[00469] Useful genomic, polynucleotide and polypeptide information about human
Mfge8 are
provided in, for example, the GeneCard Identifier GC15M088898, HGNC No. 7036,
NCBI Gene
ID 4240, Uniprot No. Q08431, and NCBI RefSeq Nos. NP 001108086.1, NM
001114614.2,
NP 001297248.1, NM 001310319.1, NP 001297249.1, NM 001310320.1, NP
001297250.1,
NM 001310321.1, NP 005919.2, and NM 005928.3.
[00470] Useful genomic, polynucleotide and polypeptide information about human
SerpinB9
are provided in, for example, the GeneCard Identifier GC06M002887, HGNC No.
8955, NCBI
Gene ID 5272, Uniprot No. P50453, and NCBI RefSeq Nos. NP 004146.1, NM
004155.5,
XP 005249241.1, and XM 005249184.4.
[00471] Methods for modulating expression of genes and factors (proteins)
include genome
editing technologies, and RNA or protein expression technologies and the like.
For all of these
technologies, well known recombinant techniques are used, to generate
recombinant nucleic
acids as outlined herein.
[00472] In some embodiments, the cells (e.g., stem cell, induced pluripotent
stem cell,
differentiated cell, hematopoietic stem cell, primary T cell or CAR-T cell)
possess genetic
modifications that inactivate the B2M and CIITA genes and express a plurality
of exogenous
polypeptides selected from the group including CD47 and DUX4, CD47 and CD24,
CD47 and
CD27, CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 and
FRA-
C, CD47 and HLA-E, CD47 and HLA-E heavy chain, CD47 and HLA-G, CD47 and PD-L1,

CD47 and ID01, CD47 and CTLA4-Ig, CD47 and Cl-Inhibitor, CD47 and IL-10, CD47
and IL-
35, CD47 and IL-39, CD47 and FasL, CD47 and CCL21, CD47 and CCL22, CD47 and
Mfge8,
and CD47 and Serpinb9, and any combination thereof. In some instances, such
cells also possess
a genetic modification that inactivates the CD142 gene.
[00473] In some instances, a gene editing system such as the CRISPR/Cas system
is used to
facilitate the insertion of tolerogenic factors, such as the tolerogenic
factors into a safe harbor or
target locus, such as the AAVS1 locus, to actively inhibit immune rejection.
In some instances,
the tolerogenic factors are inserted into a safe harbor or target locus using
an expression vector.
In some embodiments, the safe harbor or target locus is an AAVS1, CCR5, CLYBL,
ROSA26,
103
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91),
HMGB1,
ABO, REID, FUT1, or KDM5D gene locus.
1004741 In some embodiments, expression of a target gene (e.g., CD47, DUX4, or
another
tolerogenic factor gene) is increased by expression of fusion protein or a
protein complex
containing (1) a site-specific binding domain specific for the endogenous
target gene (e.g.,
CD47, DUX4, or another tolerogenic factor gene) and (2) a transcriptional
activator.
1004751 In some embodiments, the regulatory factor is comprised of a site
specific DNA-
binding nucleic acid molecule, such as a guide RNA (gRNA). In some
embodiments, the
method is achieved by site specific DNA-binding targeted proteins, such as
zinc finger proteins
(ZFP) or fusion proteins containing ZFP, which are also known as zinc finger
nucleases (ZFNs).
1004761 In some embodiments, the regulatory factor comprises a site-specific
binding domain,
such as using a DNA binding protein or DNA-binding nucleic acid, which
specifically binds to
or hybridizes to the gene at a targeted region. In some embodiments, the
provided
polynucleotides or polypeptides are coupled to or complexed with a site-
specific nuclease, such
as a modified nuclease. For example, in some embodiments, the administration
is effected using
a fusion comprising a DNA-targeting protein of a modified nuclease, such as a
mcganuclease or
an RNA-guided nuclease such as a clustered regularly interspersed short
palindromic nucleic
acid (CRISPR)-Cas system, such as CRISPR-Cas9 system. In some embodiments, the
nuclease
is modified to lack nuclease activity. In some embodiments, the modified
nuclease is a
catalytically dead dCas9.
1004771 In some embodiments, the site specific binding domain may be derived
from a
nuclease. For example, the recognition sequences of homing endonucleases and
meganucleases
such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-
PpoI, I-SceIII, I-CreI,
I-TevI, I-TevII and I-TevIII. See also U.S. Patent No. 5,420,032; U.S. Patent
No. 6,833,252;
Belfort et al. ,(1997) Nucleic Acids Res. 25:3379-3388; Duj on et al., (1989)
Gene 82:115-118;
Perler et al, (1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends
Genet. 12:224-228;
Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast et al, (1998) J. Mol.
Biol. 280:345-353
and the New England Biolabs catalogue. In addition, the DNA-binding
specificity of homing
endonucleases and meganucleases can be engineered to bind non-natural target
sites. See, for
example, Chevalier et al, (2002) Molec. Cell 10:895-905; Epinat et al, (2003)
Nucleic Acids Res.
104
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
31:2952-2962; Ashworth eta!, (2006) Nature 441 :656-659; Paques eta!, (2007)
Current Gene
Therapy 7:49-66; U.S. Patent Publication No 2007/0117128.
[00478] Zinc finger, TALE, and CRISPR system binding domains can be
"engineered" to bind
to a predetermined nucleotide sequence, for example via engineering (altering
one or more
amino acids) of the recognition helix region of a naturally occurring zinc
finger or TALE protein.
Engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are
non-naturally
occurring. Rational criteria for design include application of substitution
rules and computerized
algorithms for processing information in a database storing information of
existing ZFP and/or
TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081;
6,453,242; and
6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO

03/016496 and U.S. Publication No. 20110301073.
[00479] In some embodiments, the site-specific binding domain comprises one or
more zinc-
finger proteins (ZFPs) or domains thereof that bind to DNA in a sequence-
specific manner. A
ZFP or domain thereof is a protein or domain within a larger protein that
binds DNA in a
sequence-specific manner through one or more zinc fingers, regions of amino
acid sequence
within the binding domain whose structure is stabilized through coordination
of a zinc ion.
[00480] Among the ZFPs are artificial ZFP domains targeting specific DNA
sequences,
typically 9-18 nucleotides long, generated by assembly of individual fingers.
ZFPs include those
in which a single finger domain is approximately 30 amino acids in length and
contains an alpha
helix containing two invariant histidine residues coordinated through zinc
with two cysteines of a
single beta turn, and having two, three, four, five, or six fingers.
Generally, sequence-specificity
of a ZFP may be altered by making amino acid substitutions at the four helix
positions (-1, 2, 3
and 6) on a zinc finger recognition helix. Thus, in some embodiments, the ZFP
or ZFP-
containing molecule is non-naturally occurring, e.g., is engineered to bind to
a target site of
choice. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141;
Pabo et al. (2001)
Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-
660; Segal et al.
(2001) Curr. Opin. Biotechnol. 12:632-637; Choo etal. (2000) Curr. Opin.
Struct. Biol. 10:411-
416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558;
7,030,215;
6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S.
Patent Publication
Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by
reference in their
entireties.
105
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1004811 Many gene-specific engineered zinc fingers are available commercially.
For example,
Sangamo Biosciences (Richmond, CA, USA) has developed a platform (CompoZr) for
zinc-
finger construction in partnership with Sigma-Aldrich (St. Louis, MO, USA),
allowing
investigators to bypass zinc-finger construction and validation altogether,
and provides
specifically targeted zinc fingers for thousands of proteins (Gaj et al.,
Trends in Biotechnology,
2013, 31(7), 397-405). In some embodiments, commercially available zinc
fingers are used or
e custom designed.
1004821 In some embodiments, the site-specific binding domain comprises a
naturally occurring
or engineered (non-naturally occurring) transcription activator-like protein
(TAL) DNA binding
domain, such as in a transcription activator-like protein effector (TALE)
protein, See, e.g.,U U.S.
Patent Publication No. 20110301073, incorporated by reference in its entirety
herein.
1004831 In some embodiments, the site-specific binding domain is derived from
the
CRISPR/Cas system. In general, "CRISPR system" refers collectively to
transcripts and other
elements involved in the expression of or directing the activity of CRISPR-
associated ("Cas")
genes, including sequences encoding a Cas gene, a tracr (trans-activating
CRISPR) sequence
(e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence
(encompassing a "direct
repeat" and a tracrRNA-processed partial direct repeat in the context of an
endogenous CRISPR
system), a guide sequence (also referred to as a -spacer" in the context of an
endogenous
CRISPR system, or a "targeting sequence"), and/or other sequences and
transcripts from a
CRISPR locus.
1004841 In general, a guide sequence includes a targeting domain comprising a
polynucleotide
sequence having sufficient complementarity with a target polynucleotide
sequence to hybridize
with the target sequence and direct sequence-specific binding of the CRISPR
complex to the
target sequence. In some embodiments, the degree of complementarity between a
guide
sequence and its corresponding target sequence, when optimally aligned using a
suitable
alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%,
95%,
97.5%, 99%, or more. In some examples, the targeting domain of the gRNA is
complementary,
e.g., at least 80, 85, 90, 95, 98 or 99% complementary, e.g., fully
complementary, to the target
sequence on the target nucleic acid.
1004851 In some embodiments, the target site is upstream of a transcription
initiation site of the
target gene. In some embodiments, the target site is adjacent to a
transcription initiation site of
106
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
the gene. In some embodiments, the target site is adjacent to an RNA
polymerase pause site
downstream of a transcription initiation site of the gene
[00486] In some embodiments, the targeting domain is configured to target the
promoter region
of the target gene to promote transcription initiation, binding of one or more
transcription
enhancers or activators, and/or RNA polymerase. One or more gRNA can be used
to target the
promoter region of the gene. In some embodiments, one or more regions of the
gene can be
targeted. In certain aspects, the target sites are within 600 base pairs on
either side of a
transcription start site (TSS) of the gene.
[00487] It is within the level of a skilled artisan to design or identify a
gRNA sequence that is or
comprises a sequence targeting a gene, including the exon sequence and
sequences of regulatory
regions, including promoters and activators. A genome-wide gRNA database for
CRISPR
genome editing is publicly available, which contains exemplary single guide
RNA (sgRNA)
target sequences in constitutive exons of genes in the human genome or mouse
genome (see e.g.,
genescript.com/gRNA-database.html; see also, Sanjana et al. (2014) Nat.
Methods, 11:783-4;
www.e-crisp.org/E-CRISP/; crispr.mit.edu/). In some embodiments, the gRNA
sequence is or
comprises a sequence with minimal off-target binding to a non-target gene.
[00488] In some embodiments, the regulatory factor further comprises a
functional domain, e.g.,
a transcriptional activator.
[00489] In some embodiments, the transcriptional activator is or contains one
or more
regulatory elements, such as one or more transcriptional control elements of a
target gene,
whereby a site-specific domain as provided above is recognized to drive
expression of such gene.
In some embodiments, the transcriptional activator drives expression of the
target gene. In some
cases, the transcriptional activator, can be or contain all or a portion of an
heterologous
transactivation domain. For example, in some embodiments, the transcriptional
activator is
selected from Herpes simplex¨derived transactivation domain, Dnmt3a
methyltransferase
domain, p65, VP16, and VP64.
[00490] In some embodiments, the regulatory factor is a zinc finger
transcription factor (ZF-
TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).
[00491] In certain embodiments, the regulatory factor further comprises a
transcriptional
regulatory domain. Common domains include, e.g., transcription factor domains
(activators,
repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc,
jun, fos, myb, max,
107
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
mad, rel, ets, bcl, myb, mos family members etc.); DNA repair enzymes and
their associated
factors and modifiers; DNA rearrangement enzymes and their associated factors
and modifiers;
chromatin associated proteins and their modifiers (e.g. kinases, acetylases
and deacetylases); and
DNA modifying enzymes (e.g., methyltransferases such as members of the DNMT
family (e.g.,
DN1V1T1, DNIVIT3A, DNMT3B, DN1VIT3L, etc., topoisomerases, helicases, ligases,
kinases,
phosphatases, polymerases, endonucleases) and their associated factors and
modifiers. See, e.g.,
U.S. Publication No. 2013/0253040, incorporated by reference in its entirety
herein.
1004921 Suitable domains for achieving activation include the HSV VP 16
activation domain
(see, e.g., Hagmann et al, J. Virol. 71, 5952-5962 (1 97)) nuclear hormone
receptors (see, e.g.,
Torchia et al., Curr. Opin. Cell. Biol. 10:373-383 (1998)); the p65 subunit of
nuclear factor
kappa B (Bitko & Bank, J. Virol. 72:5610-5618 (1998) and Doyle & Hunt,
Neuroreport 8:2937-
2942 (1997)); Liu et al., Cancer Gene Ther. 5:3-28 (1998)), or artificial
chimeric functional
domains such as VP64 (Beerli et al., (1998) Proc. Natl. Acad. Sci. USA
95:14623-33), and
degron (Molinari et al., (1999) EMBO J. 18, 6439-6447). Additional exemplary
activation
domains include, Oct 1, Oct-2A, Spl, AP-2, and CTF1 (Seipel et al., EMBOJ. 11,
4961-4968
(1992) as well as p300, CBP, PCAF, SRCI PvALF, AtHD2A and ERF-2. See, for
example,
Robyr et al, (2000) Mol. Endocrinol. 14:329-347; Collingwood et al, (1999) J.
Mol. Endocrinol
23:255-275; Leo et al, (2000) Gene 245:1-11; Manteuffel-Cymborowska (1999)
Acta Biochem.
Pol. 46:77-89; McKenna et al, (1999) J. Steroid Biochem. Mol. Biol. 69:3-12;
Malik et al, (2000)
Trends Biochem. Sci. 25:277-283; and Lemon et al, (1999) Curr. Opin. Genet.
Dev. 9:499-504.
Additional exemplary activation domains include, but are not limited to,
OsGAI, HALF-I, Cl,
API, ARF-5, -6,-1, and -8, CPRFI, CPRF4, MYC-RP/GP, and TRAB1 , See, for
example,
Ogawa et al, (2000) Gene 245:21-29; Okanami et al, (1996) Genes Cells 1:87-99;
Goff et al,
(1991) Genes Dev. 5:298-309; Cho et al, (1999) Plant Mol Biol 40:419-429;
Ulmason et al,
(1999) Proc. Natl. Acad. Sci. USA 96:5844-5849; Sprenger-Haussels et al,
(2000) Plant J. 22:1-
8; Gong et al, (1999) Plant Mol. Biol. 41:33-44; and Hobo et al. , (1999)
Proc. Natl. Acad. Sci.
USA 96:15,348-15,353.
1004931 Exemplary repression domains that can be used to make genetic
repressors include, but
are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene (TIEG), v-
erbA, SID,
MBD2, MBD3, members of the DNMT family (e.g., DNIVIT1, DNMT3A, DNWIT3B,
DNIVIT3L,
etc.), Rb, and MeCP2. See, for example, Bird et al, (1999) Cell 99:451-454;
Tyler et al, (1999)
108
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
Cell 99:443-446; Knoepfler et al, (1999) Cell 99:447-450; and Robertson et al,
(2000) Nature
Genet. 25:338-342. Additional exemplary repression domains include, but are
not limited to,
ROM2 and AtHD2A. See, for example, Chem et al, (1996) Plant Cell 8:305-321;
and Wu et al,
(2000) Plant J. 22:19-27.
1004941 In some instances, the domain is involved in epigenetic regulation of
a chromosome. In
some embodiments, the domain is a histone acetyltransferase (HAT), e.g. type-
A, nuclear
localized such as MYST family members MOZ, Yb12/Sas3, MOF, and Tip60, GNAT
family
members Gcn5 or pCAF, the p300 family members CBP, p300 or Rtt109 (Bemdsen and
Denu
(2008) Curr Opin Struct Biol 18(6):682-689). In other instances the domain is
a histone
deacetylase (HD AC) such as the class I (HDAC-1, 2, 3, and 8), class II (HDAC
IIA (HDAC-4, 5,
7 and 9), HD AC JIB (HDAC 6 and 10)), class IV (HDAC-1 1), class III (also
known as sirtuins
(SIRTs); SIRT1-7) (see Mottamal et al., (2015) Molecules 20(3):3898-3941).
Another domain
that is used in some embodiments is a histone phosphorylase or kinase, where
examples include
MSK1, MSK2, ATR, ATM, DNA-PK, Bubl, VprBP, IKK-a, PKCpi, DWZip, JAK2, PKC5,
WSTF and CK2. In some embodiments, a methylation domain is used and may be
chosen from
groups such as Ezh2, PR1\'IT1/6, PR1\'IT5/7, PRMT 2/6, CARM1, sct7/9, MILL,
ALL-1, Suv 39h,
G9a, SETDB1, Ezh2, Set2, Dotl, PRWIT 1/6, PRWIT 5/7, PR-Set7 and Suv4-20h,
Domains
involved in sumoylation and biotinylation (Lys9, 13, 4, 18 and 12) may also be
used in some
embodiments (review see Kousarides (2007) Cell 128:693-705).
1004951 Fusion molecules are constructed by methods of cloning and biochemical
conjugation
that are well known to those of skill in the art. Fusion molecules comprise a
DNA-binding
domain and a functional domain (e.g., a transcriptional activation or
repression domain). Fusion
molecules also optionally comprise nuclear localization signals (such as, for
example, that from
the SV40 medium T-antigen) and epitope tags (such as, for example, FLAG and
hemagglutinin).
Fusion proteins (and nucleic acids encoding them) are designed such that the
translational
reading frame is preserved among the components of the fusion.
[00496] Fusions between a polypeptide component of a functional domain (or a
functional
fragment thereof) on the one hand, and a non-protein DNA-binding domain (e.g.,
antibiotic,
intercalator, minor groove binder, nucleic acid) on the other, are constructed
by methods of
biochemical conjugation known to those of skill in the art. See, for example,
the Pierce Chemical
Company (Rockford, IL) Catalogue. Methods and compositions for making fusions
between a
109
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
minor groove binder and a polypeptide have been described. Mapp et al, (2000)
Proc. Natl.
Acad. Sci. USA 97:3930-3935. Likewise, CRISPR/Cas TFs and nucleases comprising
a sgRNA
nucleic acid component in association with a polypeptide component function
domain are also
known to those of skill in the art and detailed herein.
1004971 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express CD47. In some
embodiments, the present
disclosure provides a method for altering a cell genome to express CD47. In
certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of CD47 into a cell line. In certain
embodiments, the at least
one ribonucleic acid or the at least one pair of ribonucleic acids is selected
from the group
consisting of SEQ ID NOS:200784-231885 of Table 29 of W02016183041, which is
herein
incorporated by reference.
1004981 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express HLA-C. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express HLA-
C. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of HLA-C into a cell line. In certain
embodiments, the at least
one ribonucleic acid or the at least one pair of ribonucleic acids is selected
from the group
consisting of SEQ ID NOS:3278-5183 of Table 10 of W02016183041, which is
herein
incorporated by reference.
1004991 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express HLA-E. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express HLA-
E. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of HLA-E into a cell line. In certain
embodiments, the at least
one ribonucleic acid or the at least one pair of ribonucleic acids is selected
from the group
consisting of SEQ ID NOS:189859-193183 of Table 19 of W02016183041, which is
herein
incorporated by reference.
110
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005001 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express HLA-F. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express I-
ILA-F. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of HLA-F into a cell line. In certain
embodiments, the at least
one ribonucleic acid or the at least one pair of iibonucleic acids is selected
from the group
consisting of SEQ ID NOS: 688808-399754 of Table 45 of W02016183041, which is
herein
incorporated by reference.
1005011 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express HLA-G. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express HLA-
G. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of HLA-G into a stem cell line. In
certain embodiments, the at
least one ribonucleic acid or the at least one pair of ribonucleic acids is
selected from the group
consisting of SEQ ID NOS:188372-189858 of Table 18 of W02016183041, which is
herein
incorporated by reference.
1005021 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express PD-Li. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express PD-
Li. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of PD-Li into a stem cell line. In
certain embodiments, the at
least one ribonucleic acid or the at least one pair of ribonucleic acids is
selected from the group
consisting of SEQ ID NOS:193184-200783 of Table 21 of W02016183041, which is
herein
incorporated by reference.
1005031 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express CTLA4-Ig. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express
CTLA4-Ig. In certain
111
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of CTLA4-Ig into a stem cell line. In
certain embodiments, the
at least one ribonucleic acid or the at least one pair of ribonucleic acids is
selected from any one
disclosed in W02016183041, including the sequence listing.
1005041 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express CT-inhibitor. In some
embodiments, the
present disclosure provides a method for altering a cell genome to express CI-
inhibitor. In certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of CI-inhibitor into a stem cell line. In
certain embodiments,
the at least one ribonucleic acid or the at least one pair of ribonucleic
acids is selected from any
one disclosed in W02016183041, including the sequence listing.
1005051 In some embodiments, the present disclosure provides a cell (e.g., a
primary T cell and
a hypoimmunogenic stem cell and derivative thereof) or population thereof
comprising a genome
in which the cell genome has been modified to express IL-35. In some
embodiments, the present
disclosure provides a method for altering a cell genome to express IL-35. In
certain
embodiments, at least one ribonucleic acid or at least one pair of ribonucleic
acids may be
utilized to facilitate the insertion of IL-35 into a stem cell line. In
certain embodiments, the at
least one ribonucleic acid or the at least one pair of ribonucleic acids is
selected from any one
disclosed in W02016183041, including the sequence listing.
1005061 In some embodiments, the tolerogenic factors are expressed in a cell
using an
expression vector. For example, the expression vector for expressing CD47 in a
cell comprises a
polynucleotide sequence encoding CD47. The expression vector can be an
inducible expression
vector. The expression vector can be a viral vector, such as but not limited
to, a lentiviral vector.
In some embodiments, the tolerogenic factors are introduced into the cells
using fusogen-
mediated delivery or a transposase system selected from the group consisting
of conditional or
inducible transposases, conditional or inducible PiggyBac transposons,
conditional or inducible
Sleeping Beauty (SB11) transposons, conditional or inducible Mosl transposons,
and conditional
or inducible To12 transposons.
1005071 In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas
system or any
of the gene editing systems described herein) is used to facilitate the
insertion of a
112
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
polynucleotide encoding a tolerogenic factor, into a genomic locus of the
hypoimmunogenic cell.
In some cases, the polynucleotide encoding the tolerogenic factor is inserted
into a safe or target
harbor locus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26,
SHS231, F3
(CD142), MICA, MICB, LRP1 (CD91), TINIGB1, ABO, RI-ID, FUT1, or KDM5D gene
locus. In
some embodiments, the polynucleotide encoding the tolerogenic factor is
inserted into a B2M
gene locus, a CIITA gene locus, a TRAC gene locus, or a TRB gene locus. In
some
embodiments, the polynucleotide encoding the tolerogenic factor is inserted
into any one of the
gene loci depicted in Tables 2-5 provided herein. In certain embodiments, the
polynucleotide
encoding the tolerogenic factor is operably linked to a promoter.
0. Protocadherin-11 Y-linked
1005081 In certain embodiments, the present technologies disclosed herein
modulate (e.g.,
reduce or eliminate) the expression of one or more Y chromosome genes by
targeting and
modulating (e.g., reducing or eliminating) expression of the Y chromosome
gene. In some
embodiments, the modulation occurs using a gene editing system (e.g.
CRISPR/Cas system). In
some embodiments, the cell has a reduced ability to induce an innate and/or
adaptive immune
response in a recipient subject.
1005091 In certain embodiments, the present technologies disclosed herein
modulate (e.g.,
reduce or eliminate) the expression of protocadherin-11 Y-linked antigen by
targeting and
modulating (e.g., reducing or eliminating) expression of the protocadherin-11
Y-linked gene,
e.g., PCDH11Y. In some embodiments, the modulation occurs using a CRISPR/Cas
system. In
some embodiments, the cell has a reduced ability to induce an immune response
in a recipient
subject.
1005101 In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of PCDHIIY gene. In some embodiments, the target polynucleotide
sequence is a
homolog of PCDH11Y gene. In some embodiments, the target polynucleotide
sequence is an
ortholog of PCDHI1Y gene.
1005111 In some embodiments, the cells described herein comprise gene
modifications at the
gene locus encoding the protocadherin-11 Y-linked antigen protein. In other
words, the cells
comprise a genetic modification at the PCDHIIY locus. In some instances, the
nucleotide
sequence encoding the protocadherin-11 Y-linked antigen protein is set forth
in RefSeq. Nos. N
113
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NM 001278619.1, NM 032971.2, NM 032972.2, NM 032973.2, or XM 017030082.1, or
in
Genbank Nos. AJ276803, AF277053, AF332216, AF332217, AJ564958, AJ564959,
AJ564960,
AJ564961, AJ564962, AJ564963, AJ564966, or AJ56496. In some instances, the
PCDH11Y
gene locus is described in NCBI Gene ID No. 83259. In certain cases, the amino
acid sequence
of protocadherin-11 Y-linked antigen is depicted as NCBI GenBank Nos.
CAC13122.1,
AAL55729.1, AAK13468.1, AAK13469.1, CAD92429.1, CAD92430.1, CAD92431.1,
CAD92432.1, CAD92433.1, CAD92434.1, CAD92437.1, or CAD92440.1. Additional
descriptions of the protocadherin-11 Y-linked antigen protein and gene locus
can be found in
Uniprot No. Q9BZA8, HGNC Ref. No. 15813, and OMIM Ref. No. 400022.
1005121 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the PCDHI1Y gene. In some
embodiments, the
genetic modification targeting the PCDH11Y gene is generated by gene editing
the PCDH11Y
gene using gene editing tools such as but not limited to CRISPR/Cas, TALE-
nucleases, zinc
finger nucleases, other viral based gene editing system, or RNA interference.
In some
embodiments, the gene editing targets the coding sequence of the PCDHI1Y gene.
In some
instances, the cells do not generate a functional PCDH11Y gene product. In the
absence of the
PCDH11Y gene product, the cells completely lack a protocadherin-11 Y-linked
antigen.
[00513] In some embodiments, a Cas9 or a Cas12a editing system is used to
target a sequence
of the PCDH11Y gene to introduce an insertion or deletion into the gene to
disrupt its function,
and in some instances, to render it inactive. In some embodiments, a single
guide RNA is used.
In some embodiments, dual guide RNAs are used. In some embodiments, any one of
the gRNA
target sequences of Table 2A or Table 2B are used. In some instances, more
than one gRNA
target sequences of Table 2A and/or Table 2B are used for gene editing. In
some embodiments, a
Cas9 editing system includes a Cas9 protein or a fragment thereof, a tracrRNA
and a crRNA. In
some embodiments, a Cas12a editing system includes a Cas12a protein or a
fragment thereof and
a crRNA.
[00514] In some embodiments, a frame-shift insertion-deletion is introduced in
any coding
sequence of the gene. In some embodiments, a modification within the UTRs,
introns, or exons
of the gene is added to disrupt the function of the PCDH11Y gene. In some
embodiments,
CRISPR/Cas editing comprising any one or more of the gRNA target sequences of
Table 2A
and/or Table 2B are utilized.
114
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005151 In some embodiments, a modification is introduced into the PCDHI1Y
gene to
inactivate the gene. In some embodiments, coding exons such as exon 1 or exon
2 or exon 3 of
the PCDH11Y gene are targeted. In some instances, a deletion is produced using
a Cas editing
system and a guide RNA target sequence targeting a sequence at the 5' of the
PCDH11Y gene
and a guide RNA target sequence to an exon such as but not limited to exon 1
or 2. In some
embodiments, a cell described herein comprises a homozygous modification of
the PCDHI IY
gene, thereby inactivating the gene.
Table 2A. Exemplary PCDH11Y gRNA target sequences ¨ Exon 1
SEQ ID NO Position Strand
Sequence PAM
1 738 1 TTTTGTTAACATGCATGTTT AGG
2 739 1 TTTGTTAACATGCATGTTTA
GGG
3 743 1 TTAACATGCATGTTTAGGGT
TGG
4 775 -1 AAGAAGAGGAGAGAGAGAAG
AGG
5 789 -1 ACAACACTGACCAAAAGAAG
AGG
6 790 1 TTCTCTCTCTCCTCTTCTTT TGG
7 804 1 TTCTTTTGGTCAGTGTTGTG CGG
8 805 1 TCTTTTGGTCAGTGTTGTGC GGG
9 842 1 TGTCACAAGTGTTTGTTGTC
CGG
10 843 1 GTCACAAGTGTTTGTTGTCC
GGG
11 850 -1 GACCGCGAAAATGTACGTCC
CGG
12 859 1 GTCCGGGACGTACATTTTCG
CGG
13 872 -1 GGAACACCACGCATACTAGC
AGG
14 877 1 CGCGGTCCTGCTAGTATGCG
TGG
15 890 1 GTATGCGTGGTGTTCCACTC
TGG
16 893 -1 TTTTCTCCTGGGCGCCAGAG
TGG
17 898 1 GGTGTTCCACTCTGGCGCCC
AGG
18 904 -1 GATGGTGTAGTTTTTCTCCT GGG
19 905 -1 GGATGGTGTAGTTTTTCTCC
TGG
20 922 -1 TTCTGGAATTTCTTCTCGGA
TGG
21 926 -1 CGTTTTCTGGAATTTCTTCT CGG
22 939 -1 TTGCCTATCAGGACGTTTTC
TGG
23 947 1 ATTCCAGAAAACGTCCTGAT
AGG
24 950 -1 CTTTCAACAAGTTGCCTATC
AGG
25 974 -1 TTGGAATCAGCGACAAGTTA
AGG
26 993 -1 GTAGTTGTCAAGGACTTGTT
TGG
27 1003 -1 GAACTGCATAGTAGTTGTCA
AGG
28 1034 1 TTCAAGCTAGTGTACAAGAC
CGG
29 1042 -1 TCGAATCAGTGGCACATCTC
CGG
115
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
30 1053 -1
TCCTCTTCAATTCGAATCAG TGG
31 1063 1
GCCACTGATTCGAATTGAAG AGG
32 1070 1
ATTCGAATTGAAGAGGATAC TGG
33 1088 1
ACTGGTGAGATCTTCACTAC CGG
34 1096 -1
CTCACGATCAATGCGAGCGC CGG
35 1121 1
GATCGTGAGAAATTATGTGC TGG
36 1131 1
AATTATGTGCTGGTATCCCA AGG
37 1132 1
ATTATGTGCTGGTATCCCAA GGG
38 1136 -1
AAAAGCAATGCTCATCCCTT GGG
39 1137 -1
TAAAAGCAATGCTCATCCCT TGG
40 1156 1
TGAGCATTGCTTTTATGAAG TGG
41 1159 1
GCATTGCTTTTATGAAGTGG AGG
42 1174 1
AGTGGAGGTTGCCATTTTGC CGG
43 1174 -1
AAATATTTCATCCGGCAAAA TGG
44 1182 -1
ACCAGTCTAAATATTTCATC CGG
45 1192 1
GCCGGATGAAATATTTAGAC TGG
46 1245 -1
ATAACTGTTGCTGGGAACAA TGG
47 1253 -1
ATATGTTGATAACTGTTGCT GGG
48 1254 -1
GATATGTTGATAACTGTTGC TGG
49 1281 -1
GAGTTTATAGCCGAGTTCTC TGG
50 1282 1
CATATCAATTCCAGAGAACT CGG
51 1312 1
CTCTAAATATACTCTCCCAG CGG
52 1316 -1
CGTCAGGATCAACAGCCGCT GGG
53 1317 -1
ACGTCAGGATCAACAGCCGC TGG
54 1331 1
GCGGCTGTTGATCCTGACGT AGG
55 1332 -1
ACTCCGTTTATGCCTACGTC AGG
56 1340 1
GATCCTGACGTAGGCATAAA CGG
Table 2B. Exemplary PCDH11Y gRNA target sequences ¨ Exon 2
SEQ ID NO Position Strand Sequence PAM
57 42149 -1
CTGGTGTTTCAATGACATCG AGG
58 42164 1
GATGTCATTGAAACACCAGA AGG
59 42168 -1 TGTGGCATCTTGTCTCCTTC TGG
60 42186 -1
TCCTTTTGAACAATCAGTTG TGG
61 42196 1
GCCACAACTGATTGTTCAAA AGG
62 42207 1
TTGTTCAAAAGGAGTTAGAT AGG
63 42208 1
TGTTCAAAAGGAGTTAGATA GGG
64 42217 1
GGAGTTAGATAGGGAAGAGA AGG
116
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
65 42232 -1 AACCTTTACTTTCATCACAT AGG
66 42241 1 TACCTATGTGATGAAAGTAA AGG
67 42251 1 ATGAAAGTAAAGGTTGAAGA TGG
68 42254 1 AAAGTAAAGGTTGAAGATGG TGG
69 42270 -1 ATAGCAGTACTGGATCTTTG AGG
70 42280 -1 TACTTGCAAAATAGCAGTAC TGG
71 42326 -1 CTGTCTCCTT A A
AGACTGGG TGG
72 42329 -1 TCTCTGTCTCCTTAAAGACT GGG
73 42330 -1 ATCTCTGTCTCCTTAAAGAC TGG
74 42331 1 TGACAACCACCCAGTCTTTA AGG
75 42366 -1 GTGCCTACAGGAGCATTTTC TGG
76 42374 1 ATACCAGAAAATGCTCCTGT AGG
77 42378 -1 TGTGTCACTGAAGTGCCTAC AGG
78 42404 -1 CTATGTCAGCATCTGTGGCA TGG
79 42409 -1 TTCACCTATGTCAGCATCTG TGG
80 42416 1 CATGCCACAGATGCTGACAT AGG
81 42436 -1 GC TGAAAGAGAAGTGGATCT
TGG
82 42443 -1 CTAGATTGCTGAAAGAGAAG TGG
83 42469 -1 AAATAATCTCCTGGCAATGT TGG
84 42471 1 ATCTAGTCTCCAACATTGCC AGG
85 42478 -1 ATTGAGGTGAAATAATCTCC TGG
86 42494 -1 TAAGTCCAGTGGTGGCATTG AGG
87 42500 1 TTTCACCTCAATGCCACCAC TGG
88 42502 -1 GAT TGTGATAAGTCCAGTGG
TGG
89 42505 -1 TTTGATTGTGATAAGTCCAG TGG
90 42526 1 TATCACAATCAAAGAAC CAC
TGG
91 42531 1 CAATCAAAGAAC CAC TGGAT
AGG
92 42531 -1 GGTGTTTCTTCCCTATCCAG TGG
93 42532 1 AATCAAAGAAC CAC TGGATA
GGG
94 42552 -1 AAAACCAGTAACTTGTGGTT TGG
95 42557 -1 TTGCCAAAACCAGTAACTTG TGG
96 42559 1 AACACCAAACCACAAGTTAC TGG
97 42565 1 AAACCACAAGTTACTGGTTT TGG
98 42575 1 TTACTGGTTTTGGCAAGTGA TGG
99 42578 1 CTGGTTTTGGCAAGTGATGG TGG
100 42597 -1 ACCAGCACCATTGCTCTTGC TGG
101 42601 1 ATTGATGCCAGCAAGAGCAA TGG
102 42607 1 GC CAGC AAGAGC AATGGTGC
TGG
103 42647 -1 TGTATCTTATGTCAATGGAT GGG
104 42648 -1 ATGTATCTTATGTCAATGGA TGG
117
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
105 42652 -1
GACGATGTATCTTATGTCAA TGG
106 42678 -1
AGAACAACTGTGTCATTGAC AGG
107 42714 -1
AGAGCAATTTTGGTGTTGAG TGG
108 42724 -1
CACAGTTATGAGAGCAATTT TGG
109 42739 1
AATTGCTCTCATAACTGTGA CGG
110 42745 1
TCTCATAACTGTGACGGATA AGG
III 42751 1
AACTGTGACGGATAAGGATG CGG
112 42761 1
GATAAGGATGCGGACCATAA TGG
113 42764 -1
AGCATGTCACCCTGCCATTA TGG
114 42765 1
AGGATGCGGACCATAATGGC AGG
115 42766 1
GGATGCGGACCATAATGGCA GGG
116 42804 -1
AATACTGGCCTTAATCTGAA AGG
117 42807 1
ATGAAATTCCTTTCAGATTA AGG
118 42819 -1
AGGAACTGATTACTGAATAC TGG
119 42835 1
ATTCAGTAATCAGTTCCTCC TGG
120 42839 -1
GATATGCTGCATTCTCCAGG AGG
121 42842 -1
CAAGATATGCTGCATTCTCC AGG
122 42874 -1
TTTAATGGCATATTCTTTTG TGG
123 42889 -1
ATCTGCAGCCAGTAATTTAA TGG
124 42892 1
AGAATATGCCATTAAATTAC TGG
125 42905 1
AAATTACTGGCTGCAGATGC TGG
126 42921 -1
ATTGCTGACTGATTCAAAGG AGG
127 42924 -1
AGCATTGCTGACTGATTCAA AGG
128 42981 -1
ACGAAAGACTGGGTGAAAAC TGG
129 42991 -1
AGAAACAGTTACGAAAGACT GGG
130 42992 -1
TAGAAACAGTTACGAAAGAC TGG
131 43017 -1
ATGCCAGGAGAGTTATTCTC AGG
132 43025 1
ATTCCTGAGAATAACTCTCC TGG
133 43032 -1
ACTTTCATCAACTGGATGCC AGG
134 43040 -1
TTGCACTTACTTTCATCAAC TGG
135 43054 1
GTTGATGAAAGTAAGTGCAA CGG
136 43067 1
AGTGCAACGGATGCAGACAG TGG
137 43068 1
GTGCAACGGATGCAGACAGT GGG
138 43080 -1
AGGTAATTGATCTCAGCATT AGG
139 43097 1
GCTGAGATCAATTACCTGCT AGG
140 43100 -1
GTGGAGC AT C AGGGC C T AGC AGG
141 43109 -1
TGAATTCAGGTGGAGCATCA GGG
142 43110 -1
CTGAATTCAGGTGGAGCATC AGG
143 43119 -1
CGATCCAGGCTGAATTCAGG TGG
144 43122 -1
CGACGATCCAGGCTGAATTC AGG
118
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
145 43126 1
TGCTCCACCTGAATTCAGCC TGG
146 43133 -1
GCATGCCTGTACGACGATCC AGG
147 43139 1
TTCAGCCTGGATCGTCGTAC AGG
148 43180 1
GAAACTAGATAGAGAAAAAG AGG
149 43204 1
TAAATATTTATTCACAATTC TGG
150 43217 1
ACAATTCTGGCAAAAGATAA TGG
151 43218 1
CAATTCTGGCAAAAGATAAT GGG
152 43219 1
AATTCTGGCAAAAGATAATG GGG
153 43233 -1
GTGACATTGCTGGTTAAGGG TGG
154 43236 -1
ACTGTGACATTGCTGGTTAA GGG
155 43237 -1
GACTGTGACATTGCTGGTTA AGG
156 43243 -1
TACAAAGACTGTGACATTGC TGG
157 43292 -1
ATTCATTGTGAGTGAAAACT GGG
158 43293 -1
TATTCATTGTGAGTGAAAAC TGG
159 43328 -1
CATGCCTTGGAAGGTTTTCT GGG
160 43329 -1
CCATGCCTTGGAAGGTTTTC TGG
161 43335 1
ATGTCCCAGAAAACCTTCCA AGG
162 43337 -1
CTACTGTACCATGCCTTGGA AGG
163 43340 1
CCAGAAAACCTTCCAAGGCA TGG
164 43341 -1
AGTCCTACTGTACCATGCCT TGG
165 43349 1
CTTCCAAGGCATGGTACAGT AGG
166 43379 1
ACTGTAACTGATCCTGATTA TGG
167 43380 -1
GCAGAATTGTCTCCATAATC AGG
168 43414 -1
GTCATCATTCTCATCTAAAA TGG
169 43441 -1
GACACCAGTTTGTGAATCAA TGG
170 43448 1
TTCACCATTGATTCACAAAC TGG
171 43466 -1
TATCAAATGAAATATTTGGT CGG
172 43470 -1
TCTCTATCAAATGAAATATT TGG
173 43513 1
ATCTTACACTTTCTATGTAA AGG
174 43519 1
CACTTTCTATGTAAAGGCTG AGG
175 43523 1
TTCTATGTAAAGGCTGAGGA TGG
176 43526 1
TATGTAAAGGCTGAGGATGG TGG
177 43561 -1
AACCACATTTATGGTTACTT TGG
178 43570 1
TGCCAAAGTAACCATAAATG TGG
179 43570 -1
ATTGACATCAACCACATTTA TGG
180 43602 -1
TAAGGAGGGACAATGAAAAC TGG
181 43616 -1
CATAAGAATAGTTGTAAGGA GGG
182 43617 -1
TCATAAGAATAGTTGTAAGG AGG
183 43620 -1
AATTCATAAGAATAGTTGTA AGG
184 43633 1
TTACAACTATTCTTATGAAT TGG
119
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
185 43650 -1
ACTGTGCCTGGATTAGTGGA CGG
186 43654 -1
GACCACTGTGCCTGGATTAG TGG
187 43655 1
GTTCTACCGTCCACTAATCC AGG
188 43662 -1
ACC TGAAAGAC CAC TGTGCC TGG
189 43663 1
GTCCACTAATCCAGGCACAG TGG
190 43672 1
TCCAGGCACAGTGGTCTTTC AGG
191 43697 1 A
TTGCTGTTGACA A TGAC AC TGG
192 43711 1
TGACACTGGCATGAATGCAG AGG
193 43730 1
GAGGTTCGTTACAGCATTGT AGG
194 43733 1
GTTC GT TAC AGCAT TGTAGG AGG
195 43772 1 TT
TGCAATC GACCAAGAAAC AGG
196 43772 -1
ATGTTATGTTGCCTGTTTCT TGG
197 43789 1
AACAGGCAACATAACATTGA TGG
198 43814 1
AAATGTGATGTTACAGAC CT TGG
199 43820 -1
CCAACACTCTGTGTAAACCA AGG
200 43831 1
CCTTGGTTTACACAGAGTGT TGG
201 43850 1
TTGGTCAAAGCTAATGACTT AGG
202 43866 -1
ACAACACTGAAGAGAGAATC AGG
203 43921 -1
TTCATTAATCAGTGTAGCAT TGG
204 43936 1
TGCTACACTGATTAATGAAC TGG
205 43968 -1
ATCTCAGTATTTGGGGTCAC TGG
206 43975 -1
ATCAGCTATCTCAGTATTTG GGG
207 43976 -1
CATCAGCTATCTCAGTATTT GGG
208 43977 -1
ACATCAGCTATCTCAGTATT TGG
209 44002 -1
GACATAGTCACTAGTTGGTG AGG
210 44007 -1
ATCTTGACATAGTCACTAGT TGG
211 44023 1
TAGTGACTATGTCAAGATCC TGG
212 44030 -1
TGCCAGCAACAGCTGCAACC AGG
213 44039 1
ATCCTGGTTGCAGCTGTTGC TGG
214 44053 -1
AATAAC TACAAC GACAGT TA TGG
215 44095 1
TGCTGTAGTAAGATGTCGCC AGG
216 44102 -1
CAGCCTTAAGGTGTGGTGCC TGG
217 44109 -1
TTCTGAGCAGCCTTAAGGTG TGG
218 44110 1
TCGCCAGGCACCACACCTTA AGG
219 44114 -1
TGTTTTTCTGAGCAGCCTTA AGG
220 44142 1
AAAACATGCAGAATTCTGAA TGG
221 44143 1
AAACATGCAGAATTCTGAAT GGG
222 44158 -1
CTGCCTGTTTTCTGGGTTTG GGG
223 44159 -1
TCTGCCTGTTTTCTGGGTTT GGG
224 44160 -1
ATCTGCCTGTTTTCTGGGTT TGG
120
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
225 44165 -1
TTATCATCTGCCTGTTTTCT GGG
226 44166 1
CTACCCCAAACCCAGAAAAC AGG
227 44166 -1
ATTATCATCTGCCTGTTTTC TGG
228 44221 -1
ATTAAGCAGCAGGTTCTTAG GGG
229 44222 -1
CATTAAGCAGCAGGTTCTTA GGG
230 44223 -1
ACATTAAGCAGCAGGTTCTT AGG
231 44231 -1
TAGTGACAACATTAAGCAGC AGG
232 44257 1
TGTCACTATTGAAGAAACTA AGG
233 44279 1
GCAGATGATGTTGACAGTGA TGG
234 44309 -1 GC
TC TTCTAGATCAATAGGA AGG
235 44313 -1
GTTTGCTCTTCTAGATCAAT AGG
236 44329 1
TGATCTAGAAGAGCAAACAA TGG
237 44330 1
GATCTAGAAGAGCAAACAAT GGG
238 44343 1
AAACAATGGGAAAGTACAAT TGG
239 44344 1
AACAATGGGAAAGTACAATT GGG
240 44364 -1
CTGTCAGGCTTGAAAGTAGT AGG
241 44379 -1
CGGGCCAAATCAGGGCTGTC AGG
242 44386 1
CAAGCCTGACAGCCCTGATT TGG
243 44387 -1
TGTAGTGTCGGGCCAAATCA GGG
244 44388 -1
TTGTAGTGTCGGGCCAAATC AGG
245 44398 -1
AGAGGCAGATTTGTAGTGTC GGG
246 44399 -1
GAGAGGCAGATTTGTAGTGT CGG
247 44416 -1
TTGGAAGGCAGGCTGTGGAG AGG
248 44421 -1
TGAATTTGGAAGGCAGGCTG TGG
249 44427 -1
TCAGGCTGAATTTGGAAGGC AGG
250 44431 -1
AGTTTCAGGCTGAATTTGGA AGG
251 44435 -1
GGGGAGTTTCAGGCTGAATT TGG
252 44445 -1
TTCAAATTCAGGGGAGTTTC AGG
253 44454 -1
ATGTGGTGCTTCAAATTCAG GGG
254 44455 -1
GATGTGGTGCTTCAAATTCA GGG
255 44456 -1
TGATGTGGTGCTTCAAATTC AGG
256 44471 -1
GAGGCAGTTCTTGGATGATG TGG
257 44480 -1
TGTTATCGAGAGGCAGTTCT TGG
258 44490 -1
GCCACAAAGGTGTTATCGAG AGG
259 44500 1
GCCTCTCGATAACACCTTTG TGG
260 44503 -1
GATAGAGTCACAGGCCACAA AGG
261 44512 -1
ACAATTGGAGATAGAGTCAC AGG
262 44527 -1
TGAACTGCTTGAGGAACAAT TGG
263 44536 -1
GTAGGGATCTGAACTGCTTG AGG
264 44553 -1
CCACAGTCAGAAACGCTGTA GGG
121
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
265 44554 -1
GCCACAGTCAGAAACGCTGT AGG
266 44564 1
CCCTACAGCGTTTCTGACTG TGG
267 44580 -1
GGTACCTCGAAGGTTGTCAC TGG
268 44587 1
CTATCCAGTGACAACCTTCG AGG
269 44590 -1
TACGGACACAGGTACCTCGA AGG
270 44601 -1
GGTCTGGTGTGTACGGACAC AGG
[00516] In some embodiments, the gRNA target sequence is to exon 1 or exon 2
of the
PCDH11Y gene. In some embodiments, the gRNA target sequence is a gRNA of Table
2A
and/or Table 2B that induces a frameshift mutation to inactivate exon 1 or
exon 2.
[00517] In some embodiments, expression of the PCDH11Y gene is partially or
fully
inactivated by an insertion or deletion within exon 1 or exon 2 of the PCDH11Y
gene.
[00518] Assays to test whether the PCDH11Y gene has been inactivated are known
and
described herein. In one embodiment, the resulting genetic modification of the
PCDH11Y gene
by PCR and the reduction of protocadherin-11 Y-linked antigen protein
expression can be
assayed by FACS analysis. In another embodiment, protocadherin-11 Y-linked
antigen protein
expression is detected using a Western blot of cells lysates probed with
antibodies to the
protocadherin-11 Y-linked antigen protein. In another embodiment, reverse
transcriptase
polymerase chain reactions (RT-PCR) are used to confirm the presence of the
inactivating
genetic modification.
P. Neuroligin-4 Y-linked
[00519] In certain embodiments, the present technologies disclosed herein
modulate (e.g.,
reduce or eliminate) the expression of neuroligin-4 Y-linked antigen by
targeting and modulating
(e.g., reducing or eliminating) expression of the neuroligin-4 Y-linked gene,
e.g., NLGN4Y. In
some embodiments, the modulation occurs using a gene editing system (e.g.
CRISPR/Cas
system). In some embodiments, the cell has a reduced ability to induce an
innate and/or adaptive
immune response in a recipient subject.
[00520] In some embodiments, the target polynucleotide sequence of the present
disclosure is a
variant of NLGN4Y gene. In some embodiments, the target polynucleotide
sequence is a
homolog of NLGN4Y gene. In some embodiments, the target polynucleotide
sequence is an
ortholog of NLGN4Y gene.
122
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005211 In some embodiments, the cells described herein comprise gene
modifications at the
gene locus encoding the neuroligin-4 Y-linked antigen protein In other words,
the cells comprise
a genetic modification at the NLGN4Y locus. In some instances, the nucleotide
sequence
encoding the neuroligin-4 Y-linked antigen protein is set forth in RefSeq.
Nos. N
NM 001164238.1, NM 001206850.1, NM 014893.4, XM 017030034.1, XM 017030035.1,
XM 017030036.1, XM 017030037.1, 3CM 017030038.1, XM 017030040.1, or
XM 017030041.1, or in Genbank Nos. AF376804, AB023168, BX537428, AC010726,
AC010879, AC010979, AC011903, BC032567, BC113525, or BC113551. In some
instances,
the NLGN4Y gene locus is described in NCBI Gene ID No. 22829. In certain
cases, the amino
acid sequence of neuroligin-4 Y-linked antigen is depicted as NCBI GenBank
Nos.
AAM46113.1, BAA76795.2, CAD97670.1, AAH32567.1, AAI13526.1, or AAI13552.1.
Additional descriptions of the neuroligin-4 Y-linked antigen protein and gene
locus can be found
in Uniprot No. Q8NFZ3, HGNC Ref. No. 15529, and OMIM Ref. No. 400028.
1005221 In some embodiments, the engineered and/or hypoimmunogenic cells
outlined herein
comprise a genetic modification targeting the NLGN4Y gene. In some
embodiments, the genetic
modification targeting the NLGN4Y gene is generated by gene editing the NLGN4Y
gene using
gene editing tools such as but not limited to CRISPR/Cas, TALE- nucleases,
zinc finger
nucleases, other viral based gene editing system, or RNA interference. In some
embodiments, the
gene editing targets the coding sequence of the NLGN4Y gene. In some
instances, the cells do
not generate a functional NLGN4Y gene product. In the absence of the NLGN4Y
gene product,
the cells completely lack a neuroligin-4 Y-linked antigen.
1005231 In some embodiments, a Cas9 or a Cas12a editing system is used to
target a sequence
of the NLGN4Y gene to introduce an insertion or deletion into the gene to
disrupt its function,
and in some instances, to render it inactive. In some embodiments, a single
guide RNA is used.
In some embodiments, dual guide RNAs are used. In some embodiments, any one of
the gRNA
target sequences of Table 3, Table 4, and/or Table 5 are used. In some
instances, more than one
gRNA target sequences of Table 3, Table 4, and/or Table 5 are used for gene
editing. In some
embodiments, a Cas9 editing system includes a Cas9 protein or a fragment
thereof, a tracrRNA
and a crRNA. In some embodiments, a Cas12a editing system includes a Cas12a
protein or a
fragment thereof and a crRNA.
123
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005241 In some embodiments, a frame-shift insertion-deletion is introduced in
any coding
sequence of the gene. In some embodiments, a modification within the UTRs,
introns, or exons
of the gene is added to disrupt the function of the NLGN4Y gene. In some
embodiments,
CRISPR/Cas editing comprising any one or more of the gRNA target sequences of
Table 3,
Table 4, and/or Table 5 are utilized.
1005251 In some embodiments, a modification is introduced into the NLGN4Y gene
to
inactivate the gene. In some embodiments, coding exons such as exon 3 or exon
4 or exon 5 of
the NLGN4Y gene are targeted. In some instances, a deletion is produced using
a Cas editing
system and a guide RNA target sequence targeting a sequence at the 5' of the
NLGN4Y gene
and a guide RNA target sequence to an exon such as but not limited to exon 3
or exon 4 or exon
5. In some embodiments, a cell described herein comprises a homozygous
modification of the
NLGN4Y gene, thereby inactivating the gene.
Table 3. Exemplary NLGN4Y gRNA target sequences ¨ Exon 3
SEQ ID NO Position Strand Sequence PAM
271 200415 -1
CCTCCATGTAAGATCCCCCA TGG
272 200423 1
TATCCATGGGGGATCTTACA TGG
273 200426 1
CCATGGGGGATCTTACATGG AGG
274 200427 1
CATGGGGGATCTTACATGGA GGG
275 200433 1
GGATCTTACATGGAGGGAAC CGG
276 200441 -1
GCTGCCATCAATCATGTTAC CGG
277 200448 1
GGAACCGGTAACATGATTGA TGG
278 200459 1
CATGATTGATGGCAGCATTT TGG
279 200469 1
GGCAGCATTTTGGCCAGCTA TGG
280 200470 1
GCAGCATTTTGGCCAGCTAT GGG
281 200471 -1
AACGATGACGTTCCCATAGC TGG
282 200498 -1
TATTCCCAGACGGTAGTTAA TGG
283 200504 1
TATCACCATTAACTACCGTC TGG
284 200505 1
ATCACCATTAACTACCGTCT GGG
Table 4. Exemplary NLGN4Y gRNA target sequences ¨ Exon 4
SEQ ID NO Position Strand Sequence PAM
285 301455 1
GTTTTTAAGTACCGGTGACC AGG
286 301455 -1
GCCTTTTGCTGCCTGGTCAC CGG
287 301462 -1
CATAGTTGCCTTTTGCTGCC TGG
288 301465 1
ACCGGTGACCAGGCAGCAAA AGG
124
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
289 301474 1
CAGGCAGCAAAAGGCAACTA TGG
290 301475 1
AGGCAGC AAAAGGC AAC TAT GGG
291 301482 1
AAAAGGCAACTATGGGCTCC TGG
292 301489 -1
TCAGTGCTTGAATCTGATCC AGG
293 301502 1
TGGATCAGATTCAAGCACTG AGG
294 301505 1
ATCAGATTCAAGCACTGAGG TGG
295 301512 1
TCAAGC ACTGAGGTGGATTG AGG
296 301522 1
AGGT GGAT T GAGGAGAAT GT CGG
297 301531 1
GAGGAGAATGTCGGAGCCTT TGG
298 301534 1
GAGAAT GTC GGAGC C TT TGG CGG
299 301535 1
AGAATGTCGGAGCCTTTGGC GGG
300 301536 1
GAATGTCGGAGCCTTTGGCG GGG
301 301536 -1
TCTCTTGGGGTCCCCGCCAA AGG
302 301549 -1
CAAAGATAGTCACTCTCTTG GGG
303 301550 -1
CCAAAGATAGTCACTCTCTT GGG
304 301551 -1
GCCAAAGATAGTCACTCTCT TGG
305 301561 1 C
CCAAGAGAGT GAC TATC TT TGG
306 301566 1
GAGAGTGACTATC TT TGGC T CGG
307 301567 1
AGAGTGAC TATC TT TGGC TC GGG
308 301568 1
GAGTGACTATCTTTGGCTCG GGG
309 301569 1
AGTGACTATCTTTGGCTCGG GGG
310 301573 1
ACTATCTTTGGCTCGGGGGC TGG
311 301574 1 C
TATC T TT GGC TCGGGGGC T GGG
312 301575 1
TATC T TT GGC TCGGGGGC T G GGG
313 301587 -1
GGTC AAC AGGC T GAC AC AGG AGG
314 301590 -1
CAGGGTCAACAGGCTGACAC AGG
315 301600 -1
AGTAGTGGGACAGGGTCAAC AGG
Table 5. Exemplary NLGN4Y gRNA target sequences ¨ Exon 5
SEQ ID NO Position Strand Sequence PAM
316 306992 -1
CGCTCTGAATGATGGCCTTC TGG
317 307000 -1
GGCAGTGC CGC TC TGAAT GA TGG
318 307004 1
CAGAAGGCCATCATTCAGAG CGG
319 307021 -1
GTTCACTGCCCAGCTGGACA GGG
320 307022 -1
AGT TC AC T GC C C AGC T GGAC AGG
321 307023 1 GC
GGCAC TGC CCTGTCCAGC TGG
322 307024 1
CGGCACTGCCCTGTCCAGCT GGG
323 307027 -1
CTGGTAGTTCACTGCCCAGC TGG
125
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
324 307042 1
CTGGGCAGTGAACTACCAGC CGG
325 307046 -1
TCCGAGTGTACTTGGCCGGC TGG
326 307050 -1
AATATCCGAGTGTACTTGGC CGG
327 307054 -1 T
GC C AATATC C GAGTGTAC T TGG
328 307056 1 AC
CAGC CGGC CAAGTACAC T CGG
329 307063 1
GGCCAAGTACACTCGGATAT TGG
330 307072 1 C
ACTCGGA T A TTGGC AGAC A AGG
331 307076 1
CGGATATTGGCAGACAAGGT CGG
332 307090 1
CAAGGTCGGCTGCAACATGC TGG
333 307099 1
CTGCAACATGCTGGACACCA CGG
334 307105 1 C
ATGC TGGAC ACC ACGGAC A TGG
335 307105 -1
CAGACATTCTACCATGTCCG TGG
336 307135 1
TCTGAAGAACAAGAACTACA AGG
337 307154 -1 T
GGC C GGGGT GATGGTC T GC TGG
338 307162 1
CATCCAGCAGACCATCACCC CGG
339 307162 -1
GT GGTAGGTGGC C GGGGTGA TGG
340 307168 -1
GGC TAT GTGGTAGGT GGCC G GGG
341 307169 -1
AGGC TAT GT GGTAGGTGGC C GGG
342 307170 -1
AAGGCTATGTGGTAGGTGGC CGG
343 307174 -1
CCCAAAGGCTATGTGGTAGG TGG
344 307177 -1
CGGCCC AAAGGC TAT GT GGT AGG
345 307181 -1
TCACCGGCCCAAAGGCTATG TGG
346 307184 1
GCCACCTACCACATAGCCTT TGG
347 307185 1 C
CACC TACCACATAGCCT TT GGG
348 307189 1
CTACCACATAGCCTTTGGGC CGG
349 307189 -1
GCCGTCGATCACCGGCCCAA AGG
350 307197 -1
ATGACGTCGCCGTCGATCAC CGG
351 307199 1 GC
C T TT G GGC C GGT GAT C GA CGG
352 307220 -1
TCAGGATCTGGGGGTCGTCT GGG
353 307221 -1
ATCAGGATCTGGGGGTCGTC TGG
354 307229 -1
CTTGCTCCATCAGGATCTGG GGG
355 307230 -1
CCTTGCTCCATCAGGATCTG GGG
356 307231 -1
GCCTTGCTCCATCAGGATCT GGG
357 307232 -1
CGCCTTGCTCCATCAGGATC TGG
358 307234 1
AGACGACCCCCAGATCCTGA TGG
359 307238 -1
GGAACTCGCCTTGCTCCATC AGG
360 307241 1
CCCCAGATCCTGATGGAGCA AGG
361 307259 -1 C
C AGC AT GAT GTC GTAGTT G AGG
362 307270 1
CCTCAACTACGACATCATGC TGG
363 307271 1
CTCAACTACGACATCATGCT GGG
126
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
364 307283 1
ATCATGCTGGGCGTCAACCA AGG
365 307284 1
TCATGCTGGGCGTCAACCAA GGG
366 307285 1
CATGCTGGGCGTCAACCAAG GGG
367 307289 1
CTGGGCGTCAACCAAGGGGA AGG
368 307289 -1
CGAACTTCAGGCCTTCCCCT TGG
369 307301 -1
CGATGCCGTCCACGAACTTC AGG
370 307303 1
AGGGGAAGGCCTGAAGTTCG TGG
371 307307 1
GAAGGCCTGAAGTTCGTGGA CGG
372 307315 1
GAAGTTCGTGGACGGCATCG TGG
373 307324 1
GGACGGCATCGTGGATAACG AGG
374 307328 1
GGCATCGTGGATAACGAGGA CGG
375 307347 -1
ACGGAGAAGTCAAAGTCGTT GGG
376 307348 -1
CACGGAGAAGTCAAAGTCGT TGG
377 307366 -1
GTTGTCCACGAAGTTGGACA CGG
378 307372 1
CTTCTCCGTGTCCAACTTCG TGG
379 307372 -1
GTAAAGGTTGTCCACGAAGT TGG
380 307385 1
AACTTCGTGGACAACCTTTA CGG
381 307388 -1
TCCCTTCAGGGTAGCCGTAA AGG
382 307397 1
AACCTTTACGGCTACCCTGA AGG
383 307398 1
ACCTTTACGGCTACCCTGAA GGG
384 307400 -1
GCAAAGTGTCTTTCCCTTCA GGG
385 307401 -1
CGCAAAGTGTCTTTCCCTTC AGG
386 307413 1
CTGAAGGGAAAGACACTTTG CGG
387 307414 1
TGAAGGGAAAGACACTTTGC GGG
388 307443 1
TCAAGTTCATGTACACAGAC TGG
389 307444 1
CAAGTTCATGTACACAGACT GGG
390 307453 1
GTACACAGACTGGGCCGATA AGG
391 307456 -1
CGTCTCCGGGTTTTCCTTAT CGG
392 307462 1
CTGGGCCGATAAGGAAAACC CGG
393 307469 -1
GGGTTTTCCGCCGCGTCTCC GGG
394 307470 1
ATAAGGAAAACCCGGAGACG CGG
395 307470 -1
AGGGTTTTCCGCCGCGTCTC CGG
396 307473 1
AGGAAAACCCGGAGACGCGG CGG
397 307483 1
GGAGACGCGGCGGAAAACCC TGG
398 307486 1
GACGCGGCGGAAAACCCTGG TGG
399 307489 -1
GTCAGTAAAGAGAGCCACCA GGG
400 307490 -1
GGTCAGTAAAGAGAGCCACC AGG
401 307509 1
CTCTCTTTACTGACCATCAG TGG
402 307510 1
TCTCTTTACTGACCATCAGT GGG
403 307511 -1
CGGCGGGGGCCACCCACTGA TGG
127
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
404 307513 1
CTTTACTGACCATCAGTGGG TGG
405 307525 1
TCAGTGGGTGGCCCCCGCCG TGG
406 307525 -1
GT C GGC GGTGGC CAC GGC GG GGG
407 307526 -1
GGTC GGC GGTGGC C AC GGC G GGG
408 307527 -1
AGGT C GGC GGT GGC C AC GGC GGG
409 307528 -1
CAGGTCGGCGGTGGCCACGG CGG
410 307531 -1
GT GC A GGTC GGCGGTGGCC A CGG
411 307537 -1 C
T GC GC GT GCAGGT C GGC GG TGG
412 307540 -1
GTACTGCGCGTGCAGGTCGG CGG
413 307543 -1
GC C GTAC T GC GC GTGC AGGT CGG
414 307547 -1
GGGAGC C GTAC T GC GC GT GC AGG
415 307553 1
GCCGACCTGCACGCGCAGTA CGG
416 307567 -1
GAAGGCATAGAAGTAGGTGG GGG
417 307568 -1
AGAAGGCATAGAAGTAGGTG GGG
418 307569 -1
TAGAAGGCATAGAAGTAGGT GGG
419 307570 -1
ATAGAAGGCATAGAAGTAGG TGG
420 307573 -1
AT GATAGAAGGCATAGAAGT AGG
421 307585 -1
GC TTT GGC AGTGAT GAT AGA AGG
422 307601 -1
AGCTGGGCTTCATTTCGCTT TGG
423 307614 1
AAAGCGAAATGAAGCCCAGC TGG
424 307615 1
AAGCGAAATGAAGCCCAGCT GGG
425 307617 -1 T
GGGC C GAAT C T GC C CAGC T GGG
426 307618 -1
AT GGGC C GAAT C T GC C C AGC TGG
427 307624 1
GAAGCCCAGCTGGGCAGATT CGG
428 307631 1
AGC TGGGCAGAT TC GGC C C A TGG
429 307636 -1
ATAGGGGAC TT CAT C GC CAT GGG
430 307637 -1 C
ATAGGGGAC T TC ATC GC CA TGG
431 307652 -1 T
GGGGAT GC C GAAGACATAG GGG
432 307653 -1
AT GGGGAT GC C GAAGAC ATA GGG
433 307654 -1
CATGGGGATGCCGAAGACAT AGG
434 307655 1
GATGAAGTCCCCTATGTC TT CGG
435 307670 1
GTCTTCGGCATCCCCATGAT CGG
436 307670 -1
GCTCTGTGGGACCGATCATG GGG
437 307671 -1
AGC TC TGT GGGAC C GAT CAT GGG
438 307672 -1
GAGCTCTGTGGGACCGATCA TGG
439 307683 -1
TTACAACTGAAGAGCTCTGT GGG
440 307684 -1
AT TAC AAC TGAAGAGC T C TG TGG
441 307711 -1
AC TGAGC AT GAC GT C GT TC T TGG
442 307729 1
CGACGTCATGCTCAGTGCCG TGG
443 307735 -1
CGTCCAGTAGGTCATCACCA CGG
128
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO Position Strand Sequence PAM
444 307743 1
GTGCCGTGGTGATGACCTAC TGG
445 307747 -1
TTTGGCGAAGTTCGTCCAGT AGG
446 307763 1
TGGACGAACTTCGCCAAAAC TGG
1005261 In some embodiments, the gRNA target sequence is to exon 1 or exon 2
of the
NLGN4Y gene. In some embodiments, the gRNA target sequence is a gRNA of Table
3, Table
4, and/or Table 5 that induces a frameshift mutation to inactivate exon 3,
exon 4, or exon 5.
1005271 In some embodiments, expression of the NLGN4Y gene is partially or
fully inactivated
by an insertion or deletion within exon 3, exon 4, or exon 5 of the NLGN4Y
gene.
1005281 Assays to test whether the NLGN4Y gene has been inactivated are known
and
described herein. In one embodiment, the resulting genetic modification of the
NLGN4Y gene
by PCR and the reduction of neuroligin-4 Y-linked antigen protein expression
can be assayed by
FACS analysis. In another embodiment, neuroligin-4 Y-linked antigen protein
expression is
detected using a Western blot of cells lysates probed with antibodies to the
neuroligin-4 Y-linked
antigen protein. In another embodiment, reverse transcriptase polymerase chain
reactions (RT-
PCR) are used to confirm the presence of the inactivating genetic
modification.
Q. Chimeric Antigen Receptors
1005291 Provided herein are hypoimmunogenic cells comprising a chimeric
antigen receptor
(CAR) In some embodiments, the CAR binds to CD19 In some embodiments, the CAR
binds
to CD20. In some embodiments, the CAR binds to CD22. In some embodiments, the
CAR
binds to CD19. In some embodiments, the CAR binds to CD19 and CD22. In some
embodiments, the CAR is selected from the group consisting of a first
generation CAR, a second
generation CAR, a third generation CAR, and a fourth generation CAR. In some
embodiments,
the CAR includes a single binding domain that binds to a single target
antigen. In some
embodiments, the CAR includes a single binding domain that binds to more than
one target
antigen, e.g., 2, 3, or more target antigens. In some embodiments, the CAR
includes two binding
domains such that each binding domain binds to a different target antigens. In
some
embodiments, the CAR includes two binding domains such that each binding
domain binds to
the same target antigen. Detailed descriptions of exemplary CARs including
CD19-specific,
CD20-specific, CD19/CD20-bispecific CARs, CD22-specific and CD19/CD22-
bispecific CARs
129
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
can be found in W02012/079000, W02016/149578 and W02020/014482, the
disclosures
including the sequence listings and figures are incorporated herein by
reference in their entirety.
In some embodiments, the CAR includes two binding domains such that each
binding domain
binds to the same target antigen.
1005301 In some embodiments, the CD19 specific CAR includes an anti-CD19
single-chain
antibody fragment (scFv), a transmembrane domain such as one derived from
human CD8a, a 4-
1BB (CD137) co-stimulatory signaling domain, and a CD3C signaling domain. In
some
embodiments, the CD20 specific CAR includes an anti-CD20 scFv, a transmembrane
domain
such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory signaling
domain, and
a CD3C signaling domain. In some embodiments, the CD19/CD20-bispecific CAR
includes an
anti-CD19 scFv, an anti-CD20 scFv, a transmembrane domain such as one derived
from human
CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3 signaling
domain. In
some embodiments, the CD22 specific CAR includes an anti-CD22 scFv, a
transmembrane
domain such as one derived from human CD8a, a 4-1BB (CD137) co-stimulatory
signaling
domain, and a CD3C signaling domain. In some embodiments, the CD19/CD22-
bispecific CAR
includes an anti-CD19 scFv, an anti-CD22 scFv, a transmembrane domain such as
one derived
from human CD8a, a 4-1BB (CD137) co-stimulatory signaling domain, and a CD3C
signaling
domain.
1005311 In some embodiments, the CAR comprises a commercial CAR construct
carried by a T
cell. Non-limiting examples of commercial CAR-T cell based therapies include
brexucabtagene
autoleucel (TECARTUSC), axicabtagene ciloleucel (YESCARTAC), idecabtagene
vicleucel
(ABECMAg), lisocabtagene maraleucel (BREYANZEC), tisagenlecleucel
(KYlVIRIAllg),
Descartes-08 and Descartes-11 from Cartesian Therapeutics, CTL119 from
Novartis, P-BMCA-
101 from Poseida Therapeutics, PBCAR19B and PBCAR269A from Precision
Biosciences,
FT819 from Fate Therapeutics, and CYAD-211 from Clyad Oncology.
1005321 In some embodiments, a hypoimmunogenic cell described herein comprises
a
polynucleotide encoding a chimeric antigen receptor (CAR) comprising an
antigen binding
domain. In some embodiments, a hypoimmunogenic cell described herein comprises
a chimeric
antigen receptor (CAR) comprising an antigen binding domain. In some
embodiments, the
polynucleotide is or comprises a chimeric antigen receptor (CAR) comprising an
antigen binding
domain. In some embodiments, the CAR is or comprises a first generation CAR
comprising an
130
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
antigen binding domain, a transmembrane domain, and at least one signaling
domain (e.g., one,
two or three signaling domains). In some embodiments, the CAR comprises a
second generation
CAR comprising an antigen binding domain, a transmembrane domain, and at least
two
signaling domains. In some embodiments, the CAR comprises a third generation
CAR
comprising an antigen binding domain, a transmembrane domain, and at least
three signaling
domains. In some embodiments, a fourth generation CAR comprising an antigen
binding
domain, a transmembrane domain, three or foul signaling domains, and a domain
which upon
successful signaling of the CAR induces expression of a cytokine gene. In some
embodiments,
the antigen binding domain is or comprises an antibody, an antibody fragment,
an scFv or a Fab.
1. Antigen binding domain (ABD) targets an antigen characteristic of a
neoplastic or cancer
cell
1005331 In some embodiments, the antigen binding domain (ABD) targets an
antigen
characteristic of a neoplastic cell. In other words, the antigen binding
domain targets an antigen
expressed by a neoplastic or cancer cell. In some embodiments, the ABD binds a
tumor
associated antigen. In some embodiments, the antigen characteristic of a
neoplastic cell (e.g.,
antigen associated with a neoplastic or cancer cell) or a tumor associated
antigen is selected from
a cell surface receptor, an ion channel-linked receptor, an enzyme-linked
receptor, a G protein-
coupled receptor, receptor tyrosine kinase, tyrosine kinase associated
receptor, receptor-like
tyrosine phosphatase, receptor serine/ threonine kinase, receptor guanylyl
cyclase, histidine
kinase associated receptor, epidermal growth factor receptors (EGFR)
(including ErbBl/EGFR,
ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), fibroblast growth factor receptors
(FGFR)
(including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF 18, and FGF21),
vascular
endothelial growth factor receptors (VEGFR) (including VEGF-A, VEGF-B, VEGF-C,
VEGF-
D, and PIGF), RET Receptor and the Eph Receptor Family (including EphAl,
EphA2, EphA3,
EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphA10, EphB1, EphB2. EphB3, EphB4,
and
EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5,
CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CC-Kb,
Bestrophins,
TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2,
NAV1.3,
NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosin-l-phosphate
receptor
(S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein,
T-cell
131
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
receptor motifs, T-cell alpha chains, T-cell 3 chains, T-cell y chains, T-cell
ö chains, CCR7,
CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25,
CD28,
CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117,
CD127, CD133, CD137 (4-1BB), CD163, F4/80, IL-4Ra, Sca-1 , CTLA-4, GITR, GARP,
LAP,
granzyme B, LFA-1, transferrin receptor, NKp46, perforin, CD4+, Thl, Th2,
Th17, Th40, Th22,
Th9, Tfh, canonical Treg. FoxP3+, Trl, Th3, Treg17, TREG; CDCP, NT5E, EpCAM,
CEA,
gpA33, mucins, TAG-72, carbonic anhydiase IX, PSMA, folate binding protein,
gangliosides
(e.g., CD2, CD3, GM2), Lewis-72, VEGF, VEGFR 1/2/3, ctV133, ct5131,
ErbBl/EGFR,
ErbB1/HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP,
Tenascin,
PDL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-113, ALK, RANKL, mTOR, CTLA-4,

IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, TIMP I, PLAUR, PTPRJ,
LTBR, ANTXR1, folate receptor alpha (FRa), ERBB2 (Her2/neu), EphA2, IL-13Ra2,
epidermal
growth factor receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30,
CD171, CS-1,
CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN,
Li-CAM, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP,
TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-
11Ra),
PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-
beta
(PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-1

receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5,

HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D,
CX0RF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2,
HAVCR1, ADRB3, PANX3, GPR20, LY6K, 0R51E2, TARP, WT1, NY-ESO-1, LAGE-la,
MAGE-AL legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-
1,
MAD-CT-2, major histocompatibility complex class I-related gene protein (MR1),
urokinase-
type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53
mutant, prostein,
survivin, telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT,
sarcoma
translocation breakpoints, ML-IAP, ERG (T1\'IPRSS2 ETS fusion gene), NA17,
PAX3, androgen
receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, 0Y-TES1,
LCK,
AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2,
intestinal
carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, L1LRA2,
CD300LF,
CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, a neoantigen, CD133, CD15,
CD184,
132
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA-A,B,C) CD49f, CD151
CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or antigenic
portion thereof.
2. ABD targets an antigen characteristic of a T cell
1005341 In some embodiments, the antigen binding domain targets an antigen
characteristic of a
T cell. In some embodiments, the ABD binds an antigen associated with a T
cell. In some
instances, such an antigen is expressed by a T cell or is located on the
surface of a T cell. In
some embodiments, the antigen characteristic of a T cell or the T cell
associated antigen is
selected from a cell surface receptor, a membrane transport protein (e.g., an
active or passive
transport protein such as, for example, an ion channel protein, a pore-forming
protein, etc.), a
transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein
characteristic of a
T cell. In some embodiments, an antigen characteristic of a T cell may be a G
protein-coupled
receptor, receptor tyrosine kinase, tyrosine kinase associated receptor,
receptor-like tyrosine
phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase,
histidine kinase
associated receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD36); CD3E
(CD3c); CD3G (CD37); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247
(CD3);
CTLA-4 (CD152); ELK1; ERK1 (MAPK3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; FILA-
DRA; HLA-DRB I; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB;
IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT; LCK; MAP2K1 (MEK1);
MAP2K2 (MEK2); MAP2K3 (MKK3); MAP2K4 (1VIKK4); MAP2K6 (MKK6); MAP2K7
(MKK7); MAP3K1 (MEKK1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14 (NIK);
MAPK8 (JNK1); MAPK9 (JNK2); MAPK10 (JNK3); MAPK11 (p383); MAPK12 (p387);
MAPK13 (p386), MAPK14 (p38a), NCK, NFAT1, NFAT2, NFKB1, NFKB2, NFKBIA,
NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA; PIK3CB;
PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (Perforin); PTEN; RAC1;
RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2;
or ZAP70.
3. ABD targets an antigen characteristic of an autoimmune diseases/disorders
and/or
inflammatory diseases/disorders
1005351 In some embodiments, the antigen binding domain targets an antigen
characteristic of
an autoimmune diseases/disorders and/or inflammatory diseases/disorder. In
some
133
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, the ABD binds an antigen associated with an autoimmune or
inflammatory
disorder. In some instances, the antigen is expressed by a cell associated
with an autoimmune
diseases/disorders and/or inflammatory diseases/disorders. In some
embodiments, the
autoimmune or inflammatory disorder is selected from chronic graft-vs-host
disease (GVHD),
lupus, arthritis, immune complex glomerulonephritis, goodpasture syndrome,
uveitis, hepatitis,
systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold
agglutinin disease,
Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia
A, Primary
Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica,
Evan's
syndrome, IgM mediated neuropathy, cryoglobulinemia, dermatomyositis,
idiopathic
thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angi
oedema, chronic
urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune
thrombocytopenia
or neutropenia or pure red cell aplasias, while exemplary non-limiting
examples of alloimmune
diseases include allosensitization (see, for example, Blazar et al., 2015, Am.
J. Transplant,
15(4):931-41) or xenosensitization from hematopoietic or solid organ
transplantation, blood
transfusions, pregnancy with fetal allosensitization, neonatal alloimmune
thrombocytopenia,
hemolytic disease of the newborn, sensitization to foreign antigens such as
can occur with
replacement of inherited or acquired deficiency disorders treated with enzyme
or protein
replacement therapy, blood products, and gene therapy. In some embodiments,
the antigen
characteristic of an autoimmune or inflammatory disorder is selected from a
cell surface
receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G
protein-coupled
receptor, receptor tyrosine kinase, tyrosine kinase associated receptor,
receptor-like tyrosine
phosphatase, receptor serine/ threonine kinase, receptor guanylyl cyclase, or
histidine kinase
associated receptor.
1005361 In some embodiments, an antigen binding domain of a CAR binds to a
ligand expressed
on B cells, plasma cells, or plasmablasts. In some embodiments, an antigen
binding domain of a
CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319,
BCMA, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5
or
CD2. See, e.g., US 2003/0077249; WO 2017/058753; WO 2017/058850; US
2021/0230245;
WO 2019/201995A1; EP 3 781 590 Al, the contents of which are herein
incorporated by
reference.
134
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
4. ABD targets an antigen characteristic of senescent cells
1005371 In some embodiments, the antigen binding domain targets an antigen
characteristic of
senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR).
In some
embodiments, the ABD binds an antigen associated with a senescent cell. In
some instances, the
antigen is expressed by a senescent cell. In some embodiments, the CAR may be
used for
treatment or prophylaxis of disorders characterized by the aberrant
accumulation of senescent
cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and
osteoarthritis.
5. ABD targets an antigen characteristic of an infectious disease
1005381 In some embodiments, the antigen binding domain targets an antigen
characteristic of
an infectious disease. In some embodiments, the ABD binds an antigen
associated with an
infectious disease. In some instances, the antigen is expressed by a cell
affected by an infectious
disease. In some embodiments, wherein the infectious disease is selected from
HIV, hepatitis B
virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 Kaposi
sarcoma-
associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1),
Merkel cell
polyomavirus (MCV), Simian virus 40 (5V40), Epstein-Barr virus, CMV, human
papillomavirus. In some embodiments, the antigen characteristic of an
infectious disease is
selected from a cell surface receptor, an ion channel-linked receptor, an
enzyme-linked receptor,
a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase
associated receptor,
receptor-like tyrosine phosphatase, receptor serine/ threonine kinase,
receptor guanylyl cyclase,
histidine kinase associated receptor, HIV Env, gp120, or CD4-induced epitope
on HIV-1 Env.
6. ABD binds to a cell surface antigen of a cell
1005391 In some embodiments, an antigen binding domain binds to a cell surface
antigen of a
cell. In some embodiments, a cell surface antigen is characteristic of (e.g.,
expressed by) a
particular or specific cell type. In some embodiments, a cell surface antigen
is characteristic of
more than one type of cell.
1005401 In some embodiments, a CAR antigen binding domain binds a cell surface
antigen
characteristic of a T cell, such as a cell surface antigen on a T cell. In
some embodiments, an
antigen characteristic of a T cell may be a cell surface receptor, a membrane
transport protein
(e.g., an active or passive transport protein such as, for example, an ion
channel protein, a pore-
135
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a
cell adhesion
protein characteristic of a T cell. In some embodiments, an antigen
characteristic of a T cell may
be a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase
associated receptor,
receptor-like tyrosine phosphatase, receptor serine/ threonine kinase,
receptor guanylyl cyclase,
or histidine kinase associated receptor.
1005411 In some embodiments, an antigen binding domain of a CAR binds a T cell
receptor. In
some embodiments, a T cell receptor may be AKTI, AKT2, AKT3, ATF2, BCL10,
CALM1,
CD3D (CD3o); CD3E (CD3E); CD3G (CD3'); CD4; CD8; CD28, CD45; CD80 (B7-1); CD86

(B7-2); CD247 (CD3); CTLA-4 (CD152); ELKI; ERKI (MAPK3); ERK2; FOS; FYN; GRAP2

(GADS); GRB2; HLA-DRA; HLA-DRBI; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS;
IKBKA (CHUK); IKBKB; IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT;
LCK; MAP2K1 (MEKI); MAP2K2 (MEK2); MAP2K3 (MKK3); MAP2K4 (MKK4); MAP2K6
(1V1KK6); MAP2K7 (1VIKK7); MAP3K1 (MEKKI); MAP3K3; MAP3K4; MAP3K5; MAP3K8;
MAP3K14 (NIX); MAPK8 (JNK1); MAPK9 (JNK2); MAPK10 (JNK3); MAPK11 (p383);
MAPK12 (p38y); MAPKI3 (p386); MAPK14 (p38a); NCK; NFAT1; NFAT2; NFKB1; NFKB2;
NFKBIA; NRAS; PAKI; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS34); PIK3CA;
PlK3CB; PIK3CD; PlK3R1; PKCA; PKCB; PKCM; PKCQ; PLCYI; PRFI (Perforin); PTEN;
RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1;
VAV2; or ZAP70.
7. Transmembrane domain
1005421 In some embodiments, the CAR transmembrane domain comprises at least a

transmembrane region of the alpha, beta or zeta chain of a T cell receptor,
CD28, CD3 epsilon,
CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134,
CD137, CD154, or functional variant thereof. In some embodiments, the
transmembrane domain
comprises at least a transmembrane region(s) of CD8a, CD8I3, 4-1BB/CD137,
CD28, CD34,
CD4, FcERIy, CD16, 0X40/CD134, CD3, CD3E, CD3y, CD3o, TCRa, TCRI3, TCK, CD32,
CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD4OL/CD154, VEGFR2,

FAS, and FGFR2B, or functional variant thereof. antigen binding domain binds
136
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
8. Signaling domain or plurality of signaling domains
1005431 In some embodiments, a CAR described herein comprises one or at least
one signaling
domain selected from one or more of B7-1/CD80; B7-2/CD86; B7-H1/PD-L1; B7-H2;
B7-H3;
B7-H4; B7-H6; B7-H7; BTLA/CD272; CD28; CTLA-4; Gi24/VISTA/B7-H5; ICOS/CD278;
PD-1; PD-L2/B7-DC; PDCD6); 4-1BB/TNFSF9/CD137; 4-1BB Ligand/TNF SF9;
BAFF/BLyS/TNFSF13B; BAFF R/TNFRSF13C; CD27/TNFRSF7; CD27 Ligand/TNFSF7;
CD30/TNFRSF8; CD30 Ligand/TNFSF8; CD40/TNFRSF5; CD40/TNFSF5; CD40
Ligand/TNFSF5; DR3/TNFRSF25; GITR/TNFRSF18; GITR Ligand/TNFSF18;
HVEM/TNFRSFI4; LIGHT/TNFSF14; Lymphotoxin-alpha/TNF-beta; 0X40/TNFRSF4; 0X40
Ligand/TNFSF4; RELT/TNFRSF19L; TACl/TNFRSF13B; TL1A/TNFSF15; TNF-alpha; TNF
RII/TNFRSF1B); 2B4/CD244/SLAMF4; BLAME/SLAMF8; CD2; CD2F-10/SLA1V1F9;
CD48/SLAMF2; CD58/LFA-3; CD84/SLAMF5; CD229/SLAMF3; CRACC/SLAMF7; NTB-
A/SLAMF6; SLAM/CD150); CD2; CD7; CD53; CD82/Kai-1; CD90/Thyl; CD96; CD160;
CD200; CD300a/LMIR1; HLA Class I; HLA-DR; Ikaros; Integrin alpha 4/CD49d;
Integrin
alpha 4 beta 1; Integrin alpha 4 beta 7/LPAM-1; LAG-3; TCL1A; TCL1B; CRTAM;
DAP12;
Dectin-1/CLEC7A; DPPIV/CD26; EphB6; TIM-1/KEVI-1/HAVCR, TIM-4; TSLP; TSLP R;
lymphocyte function associated antigen-1 (LFA-1); NKG2C, a CD3 zeta domain, an

immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB,
CD134/0X40,
CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,
CD7,
LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or functional
fragment
thereof.
1005441 In some embodiments, the at least one signaling domain comprises a CD3
zeta domain
or an immunoreceptor tyrosine-based activation motif (ITAM), or functional
variant thereof. In
other embodiments, the at least one signaling domain comprises (i) a CD3 zeta
domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof; and (ii) a
CD28 domain, or a 4-1BB domain, or functional variant thereof. In yet other
embodiments, the
at least one signaling domain comprises a (i) a CD3 zeta domain, or an
immunoreceptor tyrosine-
based activation motif (ITAM), or functional variant thereof; (ii) a CD28
domain or functional
variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional
variant thereof. In
some embodiments, the at least one signaling domain comprises a (i) a CD3 zeta
domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof; (ii) a
137
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134
domain, or
functional variant thereof and (iv) a cytokine or costimulatory ligand
transgene.
1005451 In some embodiments, the at least two signaling domains comprise a CD3
zeta domain
or an immunoreceptor tyrosine-based activation motif (ITAM), or functional
variant thereof. In
other embodiments, the at least two signaling domains comprise (i) a CD3 zeta
domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof and (ii) a
CD28 domain, or a 4-1BB domain, or functional valiant thereof. In yet oilier
embodiments, the
at least one signaling domain comprises a (i) a CD3 zeta domain, or an
immunoreceptor tyrosine-
based activation motif (ITAM), or functional variant thereof; (ii) a CD28
domain or functional
variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional
variant thereof. In
some embodiments, the at least two signaling domains comprise a (i) a CD3 zeta
domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof (ii) a
CD28 domain or functional variant thereof (iii) a 4-1BB domain, or a CD134
domain, or
functional variant thereof and (iv) a cytokine or costimulatory ligand
transgene.
1005461 In some embodiments, the at least three signaling domains comprise a
CD3 zeta
domain or an immunoreceptor tyrosinc-based activation motif (ITAM), or
functional variant
thereof. In other embodiments, the at least three signaling domains comprise
(i) a CD3 zeta
domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or
functional variant
thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant
thereof. In yet other
embodiments, the least three signaling domains comprises a (i) a CD3 zeta
domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof (ii) a
CD28 domain or functional variant thereof and (iii) a 4-1BB domain, or a CD134
domain, or
functional variant thereof. In some embodiments, the at least three signaling
domains comprise a
(i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif
(ITAM), or
functional variant thereof; (ii) a CD28 domain or functional variant thereof
(iii) a 4-1BB
domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine
or costimulatory
ligand transgene.
1005471 In some embodiments, the CAR comprises a CD3 zeta domain or an
immunoreceptor
tyrosine-based activation motif (ITAM), or functional variant thereof. In some
embodiments, the
CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based
activation motif
138
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
(ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB
domain, or functional
variant thereof.
1005481 In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof; (ii) a
CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a
CD134 domain, or
functional variant thereof.
1005491 In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof; (ii) a
CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a
4-1BB domain, or
a CD134 domain, or functional variant thereof.
1005501 In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an
immunoreceptor tyrosine-based activation motif (ITAM), or functional variant
thereof; (ii) a
CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134
domain, or
functional variant thereof; and (iv) a cytokine or costimulatory ligand
transgene.
9. Domain which upon successful signaling of the CAR induces
expression of a cytokine
gene
1005511 In some embodiments, a first, second, third, or fourth generation CAR
further
comprises a domain which upon successful signaling of the CAR induces
expression of a
cytokine gene. In some embodiments, a cytokine gene is endogenous or exogenous
to a target
cell comprising a CAR which comprises a domain which upon successful signaling
of the CAR
induces expression of a cytokine gene. In some embodiments, a cytokine gene
encodes a pro-
inflammatory cytokine. In some embodiments, a cytokine gene encodes IL-1, IL-
2, IL-9, IL-12,
IL-18, TNF, or IFN-gamma, or functional fragment thereof. In some embodiments,
a domain
which upon successful signaling of the CAR induces expression of a cytokine
gene is or
comprises a transcription factor or functional domain or fragment thereof. In
some
embodiments, a domain which upon successful signaling of the CAR induces
expression of a
cytokine gene is or comprises a transcription factor or functional domain or
fragment thereof. In
some embodiments, a transcription factor or functional domain or fragment
thereof is or
comprises a nuclear factor of activated T cells (NEAT), an NF-kB, or
functional domain or
fragment thereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells.
Biomarker Research.
139
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
5:22 (2017); WO 2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell
therapy for
tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37 (1).
[00552] In some embodiments, the CAR further comprises one or more spacers,
e.g., wherein
the spacer is a first spacer between the antigen binding domain and the
transmembrane domain.
In some embodiments, the first spacer includes at least a portion of an
immunoglobulin constant
region or variant or modified version thereof. In some embodiments, the spacer
is a second
spacer between the transmembrane domain and a signaling domain. In some
embodiments, the
second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises
glycine and serine
residues such as but not limited to glycine-serine doublets. In some
embodiments, the CAR
comprises two or more spacers, e.g., a spacer between the antigen binding
domain and the
transmembrane domain and a spacer between the transmembrane domain and a
signaling
domain.
[00553] In some embodiments, any one of the cells described herein comprises a
nucleic acid
encoding a CAR or a first generation CAR. In some embodiments, a first
generation CAR
comprises an antigen binding domain, a transmembrane domain, and signaling
domain. In some
embodiments, a signaling domain mediates downstream signaling during T cell
activation.
[00554] In some embodiments, any one of the cells described herein comprises a
nucleic acid
encoding a CAR or a second generation CAR. In some embodiments, a second
generation CAR
comprises an antigen binding domain, a transmembrane domain, and two signaling
domains. In
some embodiments, a signaling domain mediates downstream signaling during T
cell activation.
In some embodiments, a signaling domain is a costimulatory domain. In some
embodiments, a
costimulatory domain enhances cytokine production, CAR-T cell proliferation,
and/or CAR-T
cell persistence during T cell activation.
[00555] In some embodiments, any one of the cells described herein comprises a
nucleic acid
encoding a CAR or a third generation CAR. In some embodiments, a third
generation CAR
comprises an antigen binding domain, a transmembrane domain, and at least
three signaling
domains. In some embodiments, a signaling domain mediates downstream signaling
during T
cell activation. In some embodiments, a signaling domain is a costimulatory
domain. In some
embodiments, a costimulatory domain enhances cytokine production, CAR-T cell
proliferation,
and or CAR-T cell persistence during T cell activation. In some embodiments, a
third generation
140
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CAR comprises at least two costimulatory domains. In some embodiments, the at
least two
costimulatory domains are not the same.
1005561 In some embodiments, any one of the cells described herein comprises a
nucleic acid
encoding a CAR or a fourth generation CAR. In some embodiments, a fourth
generation CAR
comprises an antigen binding domain, a transmembrane domain, and at least two,
three, or four
signaling domains. In some embodiments, a signaling domain mediates downstream
signaling
during T cell activation. In some embodiments, a signaling domain is a
costimulatory domain.
In some embodiments, a costimulatory domain enhances cytokine production, CAR-
T cell
proliferation, and or CAR-T cell persistence during T cell activation.
10. ABD comprising an antibody or antigen-binding portion thereof
1005571 In some embodiments, a CAR antigen binding domain is or comprises an
antibody or
antigen-binding portion thereof. In some embodiments, a CAR antigen binding
domain is or
comprises an scFv or Fab. In some embodiments, a CAR antigen binding domain
comprises an
scFv or Fab fragment of a CD19 antibody; CD22 antibody; T-cell alpha chain
antibody; T-cell 13
chain antibody; T-cell y chain antibody; T-cell 6 chain antibody; CCR7
antibody; CD3 antibody;
CD4 antibody; CD5 antibody; CD7 antibody; CD8 antibody; CD1lb antibody; CD11c
antibody;
CDI6 antibody; CD20 antibody; CD2 1 antibody; CD25 antibody; CD28 antibody;
CD34
antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45R0 antibody; CD52

antibody; CD56 antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95
antibody;
CD117 antibody; CD127 antibody; CD133 antibody; CD137 (4-1 BB) antibody; CD163

antibody; F4/80 antibody; IL-4Ra antibody; Sca-1 antibody; CTLA-4 antibody;
GITR antibody
GARP antibody, LAP antibody, granzyme B antibody, LFA-1 antibody, MR1
antibody, uPAR
antibody; or transferrin receptor antibody.
1005581 In some embodiments, a CAR comprises a signaling domain which is a
costimulatory
domain. In some embodiments, a CAR comprises a second costimulatory domain. In
some
embodiments, a CAR comprises at least two costimulatory domains. In some
embodiments, a
CAR comprises at least three costimulatory domains. In some embodiments, a CAR
comprises a
costimulatory domain selected from one or more of CD27, CD28, 4-1BB,
CD134/0X40, CD30,
CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,
LIGHT,
NKG2C, B7-H3, a ligand that specifically binds with CD83. In some embodiments,
if a CAR
141
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
comprises two or more costimulatory domains, two costimulatory domains are
different. In
some embodiments, if a CAR comprises two or more costimulatory domains, two
costimulatory
domains are the same.
1005591 In addition to the CARs described herein, various chimeric antigen
receptors and
nucleotide sequences encoding the same are known in the art and would be
suitable for
fusosomal delivery and reprogramming of target cells in vivo and in vitro as
described herein.
See, e.g., W02013040557, W02012079000, W02016030414, Smith T, et al., Nature
Nanotechnology. 2017. DOT: 10.1038/NNAN0.2017.57, the disclosures of which are
herein
incorporated by reference.
11. Additional Descriptions of CARs
1005601 In certain embodiments, the cell may comprise an exogenous
polynucleotideencoding a
CAR. CARs (also known as chimeric immunoreceptors, chimeric T cell receptors,
or artificial T
cell receptors) are receptor proteins that have been engineered to give host
cells (e.g., T cells) the
new ability to target a specific protein. The receptors are chimeric because
they combine both
antigen-binding and T cell activating functions into a single receptor. The
polycistronic vector
of the present disclosure may be used to express one or more CARs in a host
cell (e.g., a T cell)
for use in cell-based therapies against various target antigens. The CARs
expressed by the one
or more expression cassettes may be the same or different. In these
embodiments, the CAR may
comprise an extracellular binding domain (also referred to as a "binder") that
specifically binds a
target antigen, a transmembrane domain, and an intracellular signaling domain.
In certain
embodiments, the CAR may further comprise one or more additional elements,
including one or
more signal peptides, one or more extracellular hinge domains, and/or one or
more intracellular
costimulatory domains. Domains may be directly adjacent to one another, or
there may be one
or more amino acids linking the domains. The nucleotide sequence encoding a
CAR may be
derived from a mammalian sequence, for example, a mouse sequence, a primate
sequence, a
human sequence, or combinations thereof. In the cases where the nucleotide
sequence encoding
a CAR is non-human, the sequence of the CAR may be humanized. The nucleotide
sequence
encoding a CAR may also be codon-optimized for expression in a mammalian cell,
for example,
a human cell. In any of these embodiments, the nucleotide sequence encoding a
CAR may be at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
142
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
least 97%, at least 98%, at least 99%, or 100% identical) to any of the
nucleotide sequences
disclosed herein. The sequence variations may be due to codon-optimalization,
humanization,
restriction enzyme-based cloning scars, and/or additional amino acid residues
linking the
functional domains, etc.
1005611 In certain embodiments, the CAR may comprise a signal peptide at the N-
terminus.
Non-limiting examples of signal peptides include CD8a signal peptide, IgK
signal peptide, and
granulocyte-macrophage colony-stimulating factor receptor subunit alpha
(GMCSFR-a, also
known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal
peptide, and
variants thereof, the amino acid sequences of which are provided in Table 6
below.
Table 6. Exemplary sequences of signal peptides
SEQ ID NO: Sequence
Description
6 MALPVTALLLPLALLLHAARP CD8a signal
peptide
7 METDTLLLWVLLLWVPGSTG IgK signal
peptide
8 MLLLVTSLLLCELPHPAFLLIP GMCSFR-a
(CSF2RA)
signal peptide
1005621 In certain embodiments, the extracellular binding domain of the CAR
may comprise
one or more antibodies specific to one target antigen or multiple target
antigens. The antibody
may be an antibody fragment, for example, an scFv, or a single-domain antibody
fragment, for
example, a VIM. In certain embodiments, the scFv may comprise a heavy chain
variable region
(VH) and a light chain variable region (VI) of an antibody connected by a
linker. The Vu and the
Vt, may be connected in either order, i.e., VH-linker-VL or VL-linker-VH. Non-
limiting examples
of linkers include Whitlow linker, (G4S)n (n can be a positive integer, e.g.,
1, 2, 3, 4, 5, 6, etc.)
linker, and variants thereof. In certain embodiments, the antigen may be an
antigen that is
exclusively or preferentially expressed on tumor cells, or an antigen that is
characteristic of an
autoimmune or inflammatory disease. Exemplary target antigens include, but are
not limited to,
CD5, CD19, CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, and B cell maturation
agent
(BCMA), G-protein coupled receptor family C group 5 member D (GPRC5D)
(associated with
leukemias); CS1/SLAMF7, CD38, CD138, GPRC5D, TACT, and BCMA (associated with
myelomas); CiD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA,
CSPG4, EPHA2, FAP, FRa, lL-13Ra, Mesothelin, MUC1, MUC16, and ROR1 (associated
with
143
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
solid tumors). In any of these embodiments, the extracellular binding domain
of the CAR can be
codon-optimized for expression in a host cell or have variant sequences to
increase functions of
the extracellular binding domain.
1005631 In certain embodiments, the CAR may comprise a hinge domain, also
referred to as a
spacer. The terms "hinge" and "spacer" may be used interchangeably in the
present disclosure.
Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge
domain,
IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino
acid
sequences of which are provided in Table 7 below.
Table 7. Exemplary sequences of hinge domains
SEQ ID NO: Sequence
Description
9 TTTPAPRPPTPAPTIASQPLSLRPEACRPAA CD8a, hinge domain
GGAVHTRGLDFACD
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSP CD28 hinge domain
LFPGPSKP
113 AAAIEVMYPPPYLDNEKSNGTIIHVKGKHL CD28 hinge domain
CPSPLFPGPSKP
11 ESKYGPPCPPCP
IgG4 hinge domain
12 ESKYGPPCPSCP
IgG4 hinge domain
13 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKD IgG4 hinge-CH2-CH3
TLMISRTPEVTCVVVDVSQEDPEVQFNWY domain
VDGVEVHNAKTKPREEQFNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKGLPSSIEKTIS
KAKGQPREPQVYTLPPSQEEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCS
VMHEALHNHYTQKSLSLSLGK
1005641 In certain embodiments, the transmembrane domain of the CAR may
comprise a
transmembrane region of the alpha, beta, or zeta chain of a T cell receptor,
CD28, CD3e, CD45,
CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137,
CD154, or a functional variant thereof, including the human versions of each
of these sequences.
In other embodiments, the transmembrane domain may comprise a transmembrane
region of
CD8a, CD83, 4-11111/CD137, CD28, CD34, CD4, FcERTy, CD16, 0X40/CD134, CD3C,
CD3F.,
CD31, CD36, TCRa, TCRP, TCK, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37,
CD80, CD86, CD40, CD4OL/CD154, VEGFR2, FAS, and FGFR2B, or a functional
variant
144
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
thereof, including the human versions of each of these sequences. Table 8
provides the amino
acid sequences of a few exemplary transmembrane domains.
Table 8. Exemplary sequences of transmembrane domains
SEQ ID NO: Sequence Description
14 IYIWAPLAGTCGVLLLSLVITLYC
CD8a transmembrane domain
15
FWVLVVVGGVLACYSLLVTVAFIIF CD28 transmembrane domain
WV
114
MFWVLVVVGGVLACYSLLVTVAFII CD28 transmembrane domain
FWV
1005651 In certain embodiments, the intracellular signaling domain and/or
intracellular
costimulatory domain of the CAR may comprise one or more signaling domains
selected from
B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7,
BTLA/CD272,
CD28, CTLA-4, Gi24/VISTA/B7-H5, ICOS/CD278, PD-1, PD-L2/B7-DC, PDCD6, 4-
1BB/TNFSF9/CD137, 4-1BB Ligand/TNFSF9, BAFF/BLyS/TNFSF13B, BAFF
R/TNFRSF13C, CD27/TNFRSF7, CD27 Ligand/TNFSF7, CD30/TNFRSF8, CD30
Ligand/TNFSF8, CD40/TNFRSF5, CD40/TNFSF5, CD40 Ligand/TNFSF5, DR3/TNFRSF25,
GITR/TNFRSF18, GITR Ligand/TNFSF18, HVEM/TNFRSF14, LIGHT/TNFSF14,
Lymphotoxin-alpha/INFO, 0X40/TNFRSF4, 0X40 Ligand/TNFSF4, RELT/TNFRSF19L,
TACl/TNFRSF13B, TL1A/TNFSF15, TNFa, TNF RIPTNFRSF1B, 2B4/CD244/SLAIV1F4,
BLAME/SLAMF8, CD2, CD2F-10/SLA1VIF9, CD48/SLA1VIF2, CD58/LFA-3, CD84/SLAMF5,
CD229/SLAMF3, CRACC/SLAMF7, NTB-A/SLAMF6, SLAM/CD150, CD2, CD7, CD53,
CD82/Kai-1, CD90/Thyl, CD96, CD160, CD200, CD300a/LMIR1, HLA Class I, }ILA-DR,

Ikaros, Integrin alpha 4/CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta
7/LPAM-1, LAG-
3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, TIM-1/KIM-
1/HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-
1), NKG2C,
CD3, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-
1BB,
CD134/0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1
(LFA-1),
CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and
a
functional variant thereof including the human versions of each of these
sequences. In some
embodiments, the intracellular signaling domain and/or intracellular
costimulatory domain
comprises one or more signaling domains selected from a CD3C domain, an ITAM,
a CD28
145
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
domain, 4-1BB domain, or a functional variant thereof. Table 9 provides the
amino acid
sequences of a few exemplary intracellular costimulatory and/or signaling
domains. In certain
embodiments, as in the case of tisagenlecleucel as described below, the CD3 C
signaling domain
of SEQ ID NO:18 may have a mutation, e.g., a glutamine (Q) to lysine (K)
mutation, at amino
acid position 14 (see SEQ ID NO:115).
Table 9. Exemplary sequences of intracellular costimulatory and/or signaling
domains
SEQ ID NO: Sequence
Description
16 KRGRKKLLYIFKQPFMRPVQTTQEEDG 4-1BB costimulatory domain
CSCRFPEEEEGGCEL
17 RSKRSRLLHSDYMNWITPRRPGPTRKHY CD28 costimulatory domain
QPYAPPRDFAAYRS
18 RVKFSRSADAPAYQQGQNQLYNELNL CD3 signaling domain
GRREEYDVLDKRRGRDPEMGGKPRRK
NPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGEDGLYQGLSTATKDTYDAL
HMQALPPR
115 RVKFSRSADAPAYKQGQNQLYNELNL CD3 signaling domain (with
GRREEYDVLDKRRGRDPEMGGKPRRK Q to K mutation at position 14)
NPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDAL
1-IMQALPPR
1005661 In certain embodiments where the polycistronic vector encodes two or
more CARs, the
two or more CARs may comprise the same functional domains, or one or more
different
functional domains, as described. For example, the two or more CARs may
comprise different
signal peptides, extracellular binding domains, hinge domains, transmembrane
domains,
costimulatory domains, and/or intracellular signaling domains, in order to
minimize the risk of
recombination due to sequence similarities. Or, alternatively, the two or more
CARs may
comprise the same domains. In the cases where the same domain(s) and/or
backbone are used, it
is optional to introduce codon divergence at the nucleotide sequence level to
minimize the risk of
recombination.
CD19 CAR
1005671 In some embodiments, the CAR is a CD19 CAR ("CD19-CAR"), and in these
embodiments, the polycistronic vector comprises an expression cassette that
contains a
146
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
nucleotide sequence encoding a CD19 CAR. In some embodiments, the CD19 CAR may

comprise a signal peptide, an extracellular binding domain that specifically
binds CD19, a hinge
domain, a transmembrane domain, an intracellular costimulatory domain, and/or
an intracellular
signaling domain in tandem.
1005681 In some embodiments, the signal peptide of the CD19 CAR comprises a
CD8a signal
peptide. In some embodiments, the CD8a signal peptide comprises or consists of
an amino acid
sequence set forth in SEQ ID NO.6 or an amino acid sequence that is at least
80% identical (e.g.,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO:6. In some
embodiments, the signal peptide comprises an IgK signal peptide. In some
embodiments, the
IgK signal peptide comprises or consists of an amino acid sequence set forth
in SEQ ID NO:7 or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the
signal peptide
comprises a GMCSFR-a. or CSF2RA signal peptide. In some embodiments, the
GMCSFR-a or
CSF2RA signal peptide comprises or consists of an amino acid sequence set
forth in SEQ ID
NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% identical)
to the amino acid sequence set forth in SEQ ID NO:8.
1005691 In some embodiments, the extracellular binding domain of the CD19 CAR
is specific to
CD19, for example, human CD19. The extracellular binding domain of the CD19
CAR can be
codon-optimized for expression in a host cell or to have variant sequences to
increase functions
of the extracellular binding domain. In some embodiments, the extracellular
binding domain
comprises an immunogenically active portion of an immunoglobulin molecule, for
example, an
scFv.
1005701 In some embodiments, the extracellular binding domain of the CD19 CAR
comprises
an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises
the heavy
chain variable region (VH) and the light chain variable region (VI) of FMC63
connected by a
linker. FMC63 and the derived scFv have been described in Nicholson et al.,
Mol. Immun.
34(16-17):1157-1165 (1997) and PCT Application Publication No. W02018/213337,
the entire
contents of each of which are incorporated by reference herein. In some
embodiments, the
147
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
amino acid sequences of the entire FMC63-derived scFv (also referred to as
FMC63 scFv) and
its different portions are provided in Table 10 below. In some embodiments,
the CD19-specific
scFv comprises or consists of an amino acid sequence set forth in SEQ ID
NO:19, 20, or 25, or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:19, 20, or 25. In some
embodiments, the
CD19-specific scFv may comprise one or more CDRs having amino acid sequences
set forth in
SEQ ID NOs: 21-23 and 26-28. In some embodiments, the CD19-specific scFv may
comprise a
light chain with one or more CDRs having amino acid sequences set forth in SEQ
ID NOs: 21-
23. In some embodiments, the CD19-specific scFv may comprise a heavy chain
with one or
more CDRs having amino acid sequences set forth in SEQ ID NOs: 26-28. In any
of these
embodiments, the CD19-specific scFv may comprise one or more CDRs comprising
one or more
amino acid substitutions, or comprising a sequence that is at least 80%
identical (e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical), to any of the sequences identified. In some
embodiments, the
extracellular binding domain of the CD19 CAR comprises or consists of the one
or more CDRs
as described herein.
1005711 In some embodiments, the linker linking the VH and the VT, portions of
the scFv is a
Whitlow linker having an amino acid sequence set forth in SEQ ID NO:24. In
some
embodiments, the Whitlow linker may be replaced by a different linker, for
example, a 3xG4S
linker having an amino acid sequence set forth in SEQ ID NO:30, which gives
rise to a different
FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:29. In
certain of
these embodiments, the CD19-specific scFv comprises or consists of an amino
acid sequence set
forth in SEQ ID NO:29 or an amino acid sequence that is at least 80% identical
(e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:29.
Table 10. Exemplary sequences of anti-CD19 scFv and components
SEQ ID NO: Amino Acid Sequence Description
19 DIQMTQTTSSLSASLGDRVTISCRAS Anti-CD19 FMC63 scFv
QDISKYLNWYQQKPDGTVKLLIYHT entire sequence, with
SRLHSGVPSRFSGSGSGTDYSLTISN Whitlow linker
LEQEDIATYFCQQGNTLPYTFGGGT
148
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
KLEITGSTSGSGKPGSGEGSTKGEVK
LQESGPGLVAPSQSLSVTCTVSGVSL
PDYGVSWIRQPPRKGLEWLGVIWGS
ETTYYNSALKSRLTIIKDNSKSQVFL
KMNSLQTDDTAIYYCAKHYYYGGS
YAMDYWGQGTSVTVSS
20 DIQMTQTTSSLSASLGDRVTISCRAS Anti-CD19 FMC63 scFv
QDISKYLNVVYQQKPDGTVKLLIYHT light chain variable region
SRLHSGVPSRFSGSGSGTDYSLTISN
LEQEDIATYFCQQGNTLPYTFGGGT
KLEIT
21 QDISKY Anti-CD19 FMC63
scFv
light chain CDR1
22 HTS Anti-CD19 FMC63
scFv
light chain CDR2
23 QQGNTLPYT Anti-CD19 FMC63
scFv
light chain CDR3
24 GSTSGSGKPGSGEGSTKG Whitlow linker
25 EVKLQESGPGLVAPSQSLSVTCTVS Anti-CD19 FMC63 scFv
GVSLPDYGVSWIRQPPRKGLEWLG heavy chain variable
VIWGSETTYYNSALKSRLTIIKDNSK region
SQVFLKMNSLQTDDTAIYYCAKHY
YYGGSYNIVIDYWGQGTSVTVSS
26 GVSLPDYG Anti-CD19 FMC63
scFv
heavy chain CDR1
27 IWGSETT Anti-CD19 FMC63
scFv
heavy chain CDR2
28 AKHYYYGGSYAMDY Anti-CD19 FMC63
scFv
heavy chain CDR3
29 DIQMTQTTSSLSASLGDRVTISCRAS Anti-CD19 FMC63 scFv
QDISKYLNWYQQKPDGTVKLLIYHT entire sequence, with
SRLHSGVPSRFSGSGSGTDYSLTISN 3xG4S linker
LEQEDIATYFCQQGNTLPYTFGGGT
KLEITGGGGSGGGGSGGGGSEVKLQ
ESGPGLVAPSQSLSVTCTVSGVSLPD
YGVSWIRQPPRKGLEWLGVIWGSET
TYYNSALKSRLTIIKDNSKSQVFLK
MNSLQTDDTAIYYCAKHYYYGGSY
AMIDYWGQGTSVTVSS
149
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
30 GGGGSGGGGSGGGGS 3xG4S linker
1005721 In some embodiments, the extracellular binding domain of the CD19 CAR
is derived
from an antibody specific to CD19, including, for example, SJ25C1 (Bejcek et
al., Cancer Res.
55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9):2793-2799
(1987)), 4G7
(Meeker et al., Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek
(1995)), B4
(Freedman et al., 70:418-427 (1987)), B4 TIB12b (Kansas & Tedder, J. Immunol.
147:4094-4102
(1991); Yazawa etal., Proc. Natl. Acad. Sci. USA 102:15178-15183 (2005);
Herbst et al., J.
Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Callard et al., J.
Immunology, 148(10):
2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)). In
any of
these embodiments, the extracellular binding domain of the CD19 CAR can
comprise or consist
of the V14, the Vr, and/or one or more CDRs of any of the antibodies.
1005731 In some embodiments, the hinge domain of the CD19 CAR comprises a CD8a
hinge
domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a
hinge
domain comprises or consists of an amino acid sequence set forth in SEQ ID
NO:9 or an amino
acid sequence that is at least 80% identical (e.g., at least 80%, at least
85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the amino acid
sequence set forth in SEQ ID NO:9. In some embodiments, the hinge domain
comprises a CD28
hinge domain, for example, a human CD28 hinge domain. In some embodiments, the
CD28
hinge domain comprises or consists of an amino acid sequence set forth in SEQ
ID NO:10 or an
amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the
amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the hinge
domain
comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In
some
embodiments, the IgG4 hinge domain comprises or consists of an amino acid
sequence set forth
in SEQ ID NO:11 or SEQ ID NO:12, or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:11 or
SEQ ID NO:12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-
Ch3
domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments,
the IgG4
hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth
in SEQ ID
150
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NO: fl or an amino acid sequence that is at least 80% identical (e.g., at
least 80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:13.
1005741 In some embodiments, the transmembrane domain of the CD19 CAR
comprises a
CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In
some
embodiments, the CD8a transmembrane domain comprises or consists of an amino
acid
sequence set forth in SEQ ID NO.14 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:14. In
some embodiments, the transmembrane domain comprises a CD28 transmembrane
domain, for
example, a human CD28 transmembrane domain. In some embodiments, the CD28
transmembrane domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:15 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:15.
[00575] In some embodiments, the intracellular costimulatory domain of the
CD19 CAR
comprises a 4-1BB costimulatory domain. 4-1BB, also known as CD137, transmits
a potent
costimulatory signal to T cells, promoting differentiation and enhancing long-
term survival of T
lymphocytes. In some embodiments, the 4-1BB costimulatory domain is human. In
some
embodiments, the 4-1BB costimulatory domain comprises or consists of an amino
acid sequence
set forth in SEQ ID NO: 16 or an amino acid sequence that is at least 80%
identical (e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:16.
In some
embodiments, the intracellular costimulatory domain comprises a CD28
costimulatory domain.
CD28 is another co-stimulatory molecule on T cells. In some embodiments, the
CD28
costimulatory domain is human. In some embodiments, the CD28 costimulatory
domain
comprises or consists of an amino acid sequence set forth in SEQ ID NO:17 or
an amino acid
sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at
least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to
the amino acid
sequence set forth in SEQ ID NO: 17. In some embodiments, the intracellular
costimulatory
151
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
domain of the CD19 CAR comprises a 4-1BB costimulatory domain and a CD28
costimulatory
domain as described.
1005761 In some embodiments, the intracellular signaling domain of the CD19
CAR comprises
a CD3 zeta (c) signaling domain. CD3c associates with T cell receptors (TCRs)
to produce a
signal and contains immunoreceptor tyrosine-based activation motifs (ITA_Ms).
The CD31
signaling domain refers to amino acid residues from the cytoplasmic domain of
the zeta chain
that are sufficient to functionally transmit an initial signal necessary for T
cell activation. In
some embodiments, the CD3C signaling domain is human. In some embodiments, the
CD3
signaling domain comprises or consists of an amino acid sequence set forth in
SEQ ID NO: 18 or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:18.
1005771 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD19 CAR, including, for example, a
CD19 CAR
comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:19
or SEQ ID
NO:29, the CD8a hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of
SEQ ID
NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof. In any of these
embodiments, the CD19
CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide)
as described.
1005781 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD19 CAR, including, for example, a
CD19 CAR
comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:19
or SEQ ID
NO:29, the IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD28
transmembrane
domain of SEQ ID NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the
CD3
signaling domain of SEQ ID NO:18, and/or variants (i.e., having a sequence
that is at least 80%
identical, for example, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least
97%, at least 98%, or at least 99 identical to the disclosed sequence)
thereof. In any of these
embodiments, the CD19 CAR may additionally comprise a signal peptide (e.g., a
CD8a signal
peptide) as described.
152
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005791 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD19 CAR, including, for example, a
CD19 CAR
comprising the CD19-specific scFv having sequences set forth in SEQ ID NO:19
or SEQ ID
NO:29, the CD28 hinge domain of SEQ ID NO:10, the CD28 transmembrane domain of
SEQ ID
NO:15, the CD28 costimulatory domain of SEQ ID NO:17, the CD3C signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof. In any of these
embodiments, the CD19
CAR may additionally comprise a signal peptide (e.g., a CD8a signal peptide)
as described.
1005801 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD19 CAR as set forth in SEQ ID
NO:116 or is at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide
sequence set forth in
SEQ ID NO:116 (see Table 11). The encoded CD19 CAR has a corresponding amino
acid
sequence set forth in SEQ ID NO:117 or is at least 80% identical (e.g., at
least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:117, with the
following
components: CD8a, signal peptide, FMC63 scFv (VT -Whitlow linker-VH), CD8a
hinge domain,
CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3 signaling
domain.
1005811 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a commercially available embodiment of
CD19 CAR.
Non-limiting examples of commercially available embodiments of CD19 CARs
expressed and/or
encoded by T cells include tisagenlecleucel, lisocabtagene maraleucel,
axicabtagene ciloleucel,
and brexucabtagene autoleucel.
1005821 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding tisagenlecleucel or portions thereof.
Tisagenlecleucel
comprises a CD19 CAR with the following components: CD8a signal peptide, FMC63
scFv (VL-
3xG4S linker-VH), CD8a hinge domain, CD8a transmembrane domain, 4-1BB
costimulatory
domain, and CD3 signaling domain. The nucleotide and amino acid sequence of
the CD19
CAR in tisagenlecleucel are provided in Table 11, with annotations of the
sequences provided in
Table 12.
153
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1005831 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding lisocabtagene maraleucel or portions
thereof.
Lisocabtagene maraleucel comprises a CD19 CAR with the following components:
GMCSFR-a
or CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-VH), IgG4 hinge
domain, CD28
transmembrane domain, 4-1BB costimulatory domain, and CD3C signaling domain.
The
nucleotide and amino acid sequence of the CD19 CAR in lisocabtagene maraleucel
are provided
in Table 11, with annotations of the sequences provided in Table 13.
1005841 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding axicabtagene ciloleucel or portions
thereof.
Axicabtagene ciloleucel comprises a CD19 CAR with the following components:
GMCSFR-a or
CSF2RA signal peptide, FMC63 scFv (VL-Whitlow linker-VH), CD28 hinge domain,
CD28
transmembrane domain, CD28 costimulatory domain, and CD3 signaling domain. The

nucleotide and amino acid sequence of the CD19 CAR in axicabtagene ciloleucel
are provided in
Table H, with annotations of the sequences provided in Table 14.
1005851 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding brexucabtagene autoleucel or portions
thereof
Brexucabtagene autoleucel comprises a CD19 CAR with the following components:
GMCSFR-
a signal peptide, FMC63 scFv, CD28 hinge domain, CD28 transmembrane domain,
CD28
costimulatory domain, and CD3c signaling domain.
1005861 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD19 CAR as set forth in SEQ ID NO:
31, 33, or 35,
or is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%,
at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the
nucleotide sequence set
forth in SEQ ID NO: 31, 33, or 35. The encoded CD19 CAR has a corresponding
amino acid
sequence set forth in SEQ ID NO: 32, 34, or 36, respectively, or is at least
80% identical (e.g., at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO: 32, 34, or 36,
respectively.
Table 11. Exemplary sequences of CD19 CARs
154
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Sequence
Description
116 atggccttaccagtgaccgccttgctcctgccgctggccttgctgct
Exemplary CD19
ccacgccgccaggccggacatccagatgacacagactacatcctc CAR nucleotide
cctgtctgcctctctgggagacagagtcaccatcagttgcagggca sequence
agtcaggacattagtaaatatttaaattggtatcagcagaaaccagat
ggaactgttaaactcctgatctaccatacatcaagattacactcagg
agtcccatcaaggttcagtggcagtgggtctggaacagattattctc
tcaccattagcaacctggagcaagaagatattgccacttacttagcc
aacagggtaatacgcttccgtacacgttcggaggggggaccaagc
tggagatcacaggctccacctctggatccggcaagcccggatctg
gcgagggatccaccaagggcgaggtgaaactgcaggagtcagg
acctggcctggtggcgccctcacagagcctgtccgtcacatgc act
gtctcaggggtctcattacccgactatggtgtaagctggattcgcc a
gcctccacgaaagggtctggagtggctgggagtaatatggggtag
tgaaaccacatactataattcagctctcaaatccagactgaccatcat
caaggacaactccaagagccaagttttcttaaaaatgaacagtctgc
aaactgatgacacagccatttactactgtgccaaacattattactacg
gtggtagctatgctatggactactggggccaaggaacctcagtcac
cgtctcctcaaccacgacgccagcgccgcgaccaccaacaccgg
cgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgt
gccggccagcggcggggggcgcagtgcacacgagggggctgg
acttcgcctgtgatatctacatctgggcgcccttggccgggacttgt
ggggtccttctcctgtcactggttatcaccctttactgcaaacggggc
agaaagaaactcctgtatatattcaaacaaccatttatgagaccagta
caaactactcaagaggaagatggctgtagctgccgatttccagaag
aagaagaaggaggatgtgaactgagagtgaagttcagcaggagc
gcagacgcccccgcgtaccagcagggccagaaccagctctataa
cgagctcaatctaggacgaagagaggagtacgatgttttggacaa
gagacgtggccgggaccctgagatggggggaaagccgagaag
gaagaaccctcaggaaggcctgtacaatgaactgcagaaagataa
gatggcggaggcctacagtgagattgggatgaaaggcgagcgcc
ggaggggcaaggggcacgatggcctttaccagggtctcagtaca
gccaccaaggacacctacgacgcccttcacatgcaggccctgccc
cctcgc
117 MALPVTALLLPLALLLHAARPDIQMTQ TT S Exemplary CD19
SL SASLGDRVTISCRA SQDISKYLNWYQQK CAR amino acid
PDGTVKLLIYHT SRLHSGVP SRF S GS GS GT sequence
DYSLTISNLEQEDIATYFCQQGNTLPYTFG
GGTKLEITGST SGSGKPGSGEGSTKGEVKL
QESGPGLVAP SQ SL SVTC TVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYNSAL
KSRLTIIKDNSK SQVFLKMNSLQTDDTAIY
YCAKHYYYGGSYAMDYWGQGTSVTVS ST
TTPAPRPPTPAPTIASQPL SLRPEACRPAAG
GAVHTRGLDFACDIYIWAPLAGTCGVLLL S
155
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Sequence
Description
LVITLYCKRGRKKLLYIFKQPFMRPVQTTQ
EED GC S CRFPEEEEGGCELRVKF SRSADAP
AYQQGQNQLYNELNLGRREEYD VLDKRR
GRDPEMGGKPRRKNPQEGLYNELQKDKM
AEAY SEIGMK GERRRGK GHDGL YQGL S TA
TKDTYDALHMQALPPR
31 atggccttaccagtgaccgccttgctcctgccgctggccttgctgct
Tisagenlecleucel
ccacgccgccaggccggacatccagatgacacagactacatcctc CD19 CAR
cctgtctgcctctctgggagacagagtcaccatcagttgcagggca nucleotide
agtcaggacattagtaaatatttaaattggtatcagcagaaaccagat sequence
ggaactgttaaactcctgatctaccatacatcaagattacactcagg
agtcccatcaaggttcagtggcagtgggtctggaacagattattctc
tcaccattagcaacctggagcaagaagatattgccacttacattgcc
aacagggtaatacgcttccgtacacgttcggaggggggaccaagc
tggagatcacaggtggcggtggctcgggcggtggtgggtcgggt
ggcggcggatctgaggtgaaactgcaggagtcaggacctggcct
ggtggcgccctcacagagcctgtccgtcacatgcactgtctcagg
ggtctcattacccgactatggtgtaagctggattcgccagcctccac
gaaagggtctggagtggctgggagtaatatggggtagtgaaacca
catactataattcagctctcaaatccagactgaccatcatcaaggac
aactccaagagccaagttttcttaaaaatgaacagtctgcaaactga
tgacacagccatttactactgtgccaaacattattactacggtggtag
ctatgctatggactactggggccaaggaacctcagtcaccgtctcct
caaccacgacgccagcgccgcgaccaccaacaccggcgcccac
catcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggc
cagcggcggggggcgcagtgcacacgagggggctggacttcgc
ctgtgatatctacatctgggcgcccttggccgggacttgtggggtcc
ttctcctgtcactggttatcaccctttactgcaaacggggcagaaag
aaactcctgtatatattcaaacaaccatttatgagaccagtacaaact
actcaagaggaagatggctgtagctgccgatttccagaagaagaa
gaaggaggatgtgaactgagagtgaagttcagcaggagcgcaga
cgcccccgc,Ttacaagcagggccagaaccagctctataacgagc
tcaatctaggacgaagagaggagtacgatgttttggacaagagac
gtggccgggaccctgagatggggggaaagccgagaaggaaga
accctcaggaaggcctgtacaatgaactgcagaaagataagatgg
cggaggcctacagtgagattgggatgaaaggcgagcgccggag
gggcaaggggcacgatggcctttaccagggtctcagtacagccac
caaggacacctacgacgcccttcacatgcaggccctgccccctcg
32 MALPVTALLLPLALLLHA ARPDIQMTQTTS Ti sageni ecl eucel
SL SASLGDRVTISCRASQDISKYLNWYQQK CD19 CAR amino
PDGTVKLLIYHT SRLHSGVP SRF SGS GS GT acid sequence
DYSLTISNLEQEDIATYFCQQGNTLPYTFG
GGTKLEITGGGGSGGGGSGGGGSEVKLQE
156
CA 03225283 2024- 1- 8

9 -1 -17Z0Z E9ZSZZ0
LSI
Evv00000SpooEvoEluovoEl000SouSouloomEERB
DoE330a3E3o101,33200u3TETOTD3033uSbuo30001E30
gFEo5EaSoguEoFFERBETuo5FoTauFoguouTooSau
53351.uu'uouftuu5uoTou'uou'ulT121335ftuuoo
000gaguSSoSSopogugo0FoOSSTagST000vOSSooF
5t
D5Rel:e501.0312aaarTgata55Ruguolo
aneFinffeFarromEinFroweffeooRFFroffroorpoFino
000'uo oo guu fto oi12-eui21.ou'ai2Tua
EuvEuvaeuaeuaeoomuEooEToguTEToEETegeuEEpEup
opulaeuuoulaBoougaluTiluomumuuolluTuTuTWToolo
uvuSuRuguoSSISFouRaTSSFTTnop onooSFTFoouo
TfiEloglooffgouTo`liToofiElofi'T.Sofi'Sagolfi'FlfiETFfi'lofi.
iSROpii2lepooRn000000Si000Roov2SoviReviolua
oFuoEuFTFoouS)235uomo5FFuoogSFFTomouFFIToo
S'appS'EooSS'omaelomapoSEEooS'oRTDETomowooR
oaeouFauFoouguogTooFuouuSieFuuSToollFTEguooau
ftuo5uou'uou5ftuoluowoouT000au5uu5Toopo5
uovuouToupogooauFoSuo055gpTuFTS'oFFFToSSITv
I.005'nu55u0000005P05ooTu5i.oft5T5o5oulo
EF000SpogeSTASoESogu,STEoouoSToouETEogarogu
guooguoppooggi2gpoggpoog og-e-euggeogTogyai.
55u0o5E5ReoouoSuo555u535535u355TooSuuo5535
poSSoopoupFuoFFoouomuFFloauvvaguSSoFFoSS
TiloououT000Fiopououuo5FSuoguooFiluouToouooFoT
nuEvEEEvovvEEToovuoololvoovElooEvomovEoovog
goopOODOuDOSbOulliOS'apapaa042agOo2uoup0p0
go oguoououoouToTaTogioauu DTSbouogSbuSboogup
a ouanbas 5uo5u oluT5it,ouu5To ouTguvo 5v owaage ooguoo555
ap TO ionu 0301.35-eowoaeFT50033-
e03050TooRe3353RaTooge0
61 Go loom resew oToouoaegu000u5Tuguoowou5000pTuTofTouToo5oo
auaffeTqcoos
0000SToffeS'oRTFToFioin offroarFTFFToToFioSie
wacrIvOiAar-wcuu
mitvisloOKIDCFFIDNONITIMNIAIDIJS A
VaVIADIGNOIHNAIDHOdN)11111d)1991Alad
CEHDIRDICHACEAIMfilDINTINIAIONO 90
NAVdViaVSWS dNAW-11399HaalcIDI3S 39
GHHO,LIOAcTITIALicIoNdIATINNIIDIDIDAII
MAD AL D VIcIVMIAICD V JCIIDILLH
AVDOVVaDVaarIS IcIOS VIIdVdidaidV
cIIIIS SAIASIOOOMAGIIAIVASOOAAAHN
VOA AI-VICE/at NTAINTAA S NS
NCENTIET
)1SNIVSNAA.LiasoMIADIAMD)Ridd0)11
ANSADACHISAD SAI DIAS -IS OS dVAIDd-DS
uopdpasaa aauanbas Oas
tL,890/ZZOZSf1/Id L,Z8L,8Z/2OZ OAA

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Sequence
Description
34 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTS Lisocabtagene
SLSASLGDRVTISCRASQDISKYLNWYQQK maraleucel CD19
PDGTVKLLIYHTSRLHSGVPSRFSGSGSGT CAR amino acid
DYSLTISNLEQEDIATYFCQQGNTLPYTFG sequence
GGTKLEITGSTSGSGKPGSGEGSTKGEVKL
QESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYNSAL
KSRLTIIKDNSKSQVFLKMNSLQTDDTAIY
YCAKHYYYGGSYAMDYWGQGTSVTVSSE
SKYGPPCPPCPMFWVLVVVGGVLACYSLL
VTVAFIIFWVKRGRKKLLYIFKQPFMRPVQ
TTQEEDGCSCRFPEEEEGGCELRVKF SRSA
DAPAYQQGQNQLYNELNLGRREEYDVLD
KRRGRDPEMGGKPRRKNPQEGLYNELQK
DKMAEAYSEIGMKGERRRGKGHDGLYQG
LSTATKDTYDALHMQALPPR
35 atgcttctcctggtgacaagccttctgctctgtgagttaccacaccca
Axicabtagene
gcattectectgatcccagacatccagatgacacagactacatcctc ciloleucel CD19
cctgtctgcctctctgggagacagagtcaccatcagttgcagggca CAR nucleotide
agtcaggacattagtaaatatttaaattggtatcagcagaaaccagat sequence
ggaactgttaaactcctgatctaccatacatcaagattacactcagg
agtcccatcaaggttcagtggcagtgggtctggaacagattattctc
tcaccattagcaacctggagcaagaagatattgccacttacttagcc
aacagggtaatacgcttccgtacacgttcggaggggggactaagtt
ggaaataacaggctccacctctggatccggcaagcccggatctgg
cgagggatccaccaagggcgaggtgaaactgcaggagtcagga
cctggcctggtggcgccctcacagagcctgtccgtcacatgcactg
tctcaggggtctcattacccgactatggtgtaagctggattcgccag
cctccacgaaagggtctggagtggctgggagtaatatggggtagt
gaaaccacatactataattcagctctcaaatccagactgaccatcatc
aaggacaactccaagagccaagttttcttaaaaatgaacagtctgca
aactgatgacacagccatttactactgtgccaaacattattactacgg
tggtagctatgctatggactactggggtcaaggaacctcagtcacc
gtctcctcageggccgcaattgaagttatgtatcctcctecttaccta
gacaatgagaagagcaatggaaccattatccatgtgaaagggaaa
cacctttgtccaagtcccctatttcccggaccttctaagcccttttggg
tgctggtggtggttgggggagtcctggcttgctatagcttgctagta
acagtggcctttattattttctgggtgaggagtaagaggagcaggct
cctgcacagtgactacatgaacatgactccccgccgccccgggcc
cacccgcaagcattaccagccctatgccccaccacgcgacttcgc
agcctatcgctccagagtgaagttcagcaggagcgcagacgccc
ccgcgtaccagcagggccagaaccagctctataacgagctcaatc
taggacgaagagaggagtacgatgttttggacaagagacgtggcc
gggaccctgagatggggggaaagccgagaaggaagaaccctca
158
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Sequence
Description
ggaaggcctgtacaatgaactscagaaagataagatggeggagg
cctacagtgagattgggatgaaaggcgagcgccggaggggcaa
ggggcacgatggcctttaccagggtctcagtacagccaccaagga
cacctacgacgcccttcacatgcaggccctgccccctcgc
36 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTS Axicabtagene
SL S A SL GDRVTI SCRA S QDI SKYLNWYQ QK ciloleucel CD19
PDGTVKLLIYHTSRLHSGVPSRFSGSGSGT CAR amino acid
DYSLTISNLEQEDIATYFCQQGNTLPYTFG sequence
GGTKLEITGSTSGSGKPGSGEGSTKGEVKL
QESGPGLVAPSQSLSVTCTVSGVSLPDYGV
SWIRQPPRKGLEWLGVIWGSETTYYNSAL
KSRLTIIKDNSKSQVFLKMNSLQTDDTAIY
YCAKHYYYGGSYAMDYWGQGTSVTVSSA
AAIEVMYPPPYLDNEKSNGTIIHVKGKHLC
PSPLFPGPSKPFWVLVVVGGVLACYSLLVT
VAFIIFWVRSKRSRLLHSDYM_NMTPRRPGP
TRKHYQPYAPPRDFAAYRSRVKFSRSADA
PAYQQGQNQLYNELNLGRREEYDVLDKR
RGRDPEMGGKPRRKNPQEGLYNELQKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLS
TATKDTYDALHMQALPPR
Table 12. Annotation of tisagenlecleucel CD19 CAR sequences
Feature Nucleotide Sequence Amino Acid
Sequence
Position Position
CD8a signal peptide 1-63 1-21
FMC63 scFv 64-789 22-263
(VL-3xG4S linker-VH)
CD8a hinge domain 790-924 264-308
CD8a transmembrane domain 925-996 309-332
4-1BB costimulatory domain 997-1122 333-374
CD3C signaling domain 1123-1458 375-486
Table 13. Annotation of lisocabtagene maraleucel CD19 CAR sequences
Feature Nucleotide Sequence Amino Acid
Sequence
Position Position
GMCSFR-a signal peptide 1-66 1-22
FMC63 scFv 67-801 23-267
159
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
(VL-Whitlow linker-VH)
IgG4 hinge domain 802-837 268-279
CD28 transmembrane domain 838-921 280-307
4-1BB costimulatory domain 922-1047 308-349
CD3t signaling domain 1048-1383 350-461
Table 14. Annotation of axicabtagene ciloleucel CD19 CAR sequences
Feature Nucleotide Sequence Amino Acid
Sequence
Position Position
CSF2RA signal peptide 1-66 1-22
FMC63 scFv 67-801 23-267
(VL-Whitlow linker-VH)
CD28 hinge domain 802-927 268-309
CD28 transmembrane domain 928-1008 310-336
CD28 costimulatory domain 1009-1131 337-377
CD3 signaling domain 1132-1467 378-489
1005871 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding CD19 CAR as set forth in SEQ ID NO:
31, 33, or 35, or
at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide
sequence set forth in
SEQ ID NO: 31, 33, or 35. The encoded CD19 CAR has a corresponding amino acid
sequence
set forth in SEQ ID NO: 32, 34, or 36, respectively, is at least 80% identical
(e.g., at least 80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, at least 99%, or
100% identical) to the amino acid sequence set forth in SEQ ID NO: 32, 34, or
36, respectively.
CD20 CAR
1005881 In some embodiments, the CAR is a CD20 CAR ("CD2O-CAR"), and in these
embodiments, the polycistronic vector comprises an expression cassette that
contains a
nucleotide sequence encoding a CD20 CAR. CD20 is an antigen found on the
surface of B cells
as early at the pro-B phase and progressively at increasing levels until B
cell maturity, as well as
on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes
found in cases of
Hodgkins disease, myeloma, and thymoma. In some embodiments, the CD20 CAR may
160
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
comprise a signal peptide, an extracellular binding domain that specifically
binds CD20, a hinge
domain, a transmembrane domain, an intracellular costimulatory domain, and/or
an intracellular
signaling domain in tandem.
1005891 In some embodiments, the signal peptide of the CD20 CAR comprises a
CD8a signal
peptide. In some embodiments, the CD8a signal peptide comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:6 or an amino acid sequence that is at least
80% identical (e.g.,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO:6. In some
embodiments, the signal peptide comprises an IgK signal peptide. In some
embodiments, the
IgK signal peptide comprises or consists of an amino acid sequence set forth
in SEQ ID NO:7 or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the
signal peptide
comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-
a or
CSF2RA signal peptide comprises or consists of an amino acid sequence set
forth in SEQ ID
NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% identical)
to the amino acid sequence set forth in SEQ ID NO:8.
1005901 In some embodiments, the extracellular binding domain of the CD20 CAR
is specific to
CD20, for example, human CD20. The extracellular binding domain of the CD20
CAR can be
codon-optimized for expression in a host cell or to have variant sequences to
increase functions
of the extracellular binding domain. In some embodiments, the extracellular
binding domain
comprises an immunogenically active portion of an immunoglobulin molecule, for
example, an
scFv.
1005911 In some embodiments, the extracellular binding domain of the CD20 CAR
is derived
from an antibody specific to CD20, including, for example, Leu16, IF5, 1.5.3,
rituximab,
obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab,
ublituximab, and ocrelizumab. In some embodiments, the CD20 CAR is derived
from a CAR
specific to CD20, including, for example, MB-106, UCART20, or C-CAR066, as
detailed in
Table 15A. In any of these embodiments, the extracellular binding domain of
the CD20 CAR
161
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
can comprise or consist of the VH, the Vr, and/or one or more CDRs of any of
the antibodies or
CARs detailed in Table 15A.
Table 15A. Exemplary CD20 -specific CARs
CAR Name Antigen Company
Reference
MB-106 CD20 Fred Hutchinson Cancer
Shadman et al., Blood 134
Research Center (Suppl.
1):3235 (2019)
UCART20 CD20 Cell ecti s
www.cellbiomedgroup.com
C-CAR066 CD20 Cellular Biomedicine Group
Liang et al., J Clin Oncol
39(15) supp1:2508-2508
(2021)
1005921 In some embodiments, the extracellular binding domain of the CD20 CAR
comprises
an scFv derived from the Leu16 monoclonal antibody, which comprises the heavy
chain variable
region (Vii) and the light chain variable region (VI) of Leul6 connected by a
linker. See Wu et
al., Protein Engineering. 14(12):1025-1033 (2001). In some embodiments, the
linker is a 3xG4S
linker. In other embodiments, the linker is a Whitlow linker as described
herein. In some
embodiments, the amino acid sequences of different portions of the entire
Leu16-derived scFv
(also referred to as Leul 6 scFv) and its different portions are provided in
Table 15B below. In
some embodiments, the CD20-specific scFv comprises or consists of an amino
acid sequence set
forth in SEQ ID NO:37, 38, or 42, or an amino acid sequence that is at least
80% identical (e.g.,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO:37, 38, or 42.
In some embodiments, the CD20-specific scFv may comprise one or more CDRs
having amino
acid sequences set forth in SEQ ID NOs: 39-41, 43 and 44. In some embodiments,
the CD20-
specific scFv may comprise a light chain with one or more CDRs having amino
acid sequences
set forth in SEQ ID NOs: 39-41. In some embodiments, the CD20-specific scFv
may comprise a
heavy chain with one or more CDRs having amino acid sequences set forth in SEQ
ID NOs: 43-
44. In any of these embodiments, the CD20-specific scFv may comprise one or
more CDRs
comprising one or more amino acid substitutions, or comprising a sequence that
is at least 80%
identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at least 97%,
162
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
at least 98%, at least 99%, or 100% identical), to any of the sequences
identified. In some
embodiments, the extracellular binding domain of the CD20 CAR comprises or
consists of the
one or more CDRs as described herein.
Table 15B. Exemplary sequences of anti-CD20 scFv and components
SEQ ID NO: Amino Acid Sequence Description
37 DIVLTQSPAILSASPGEKVTMTCRAS Anti-CD20 Leu16 scFv
SSVNYMDWYQKKPGSSPKPWIYAT entire sequence, with
SNLASGVPARFSGSGSGTSYSLTISR Whitlow linker
VEAEDAATYYCQQWSFNPPTFGGG
TKLEIKGSTSGSGKPGSGEGSTKGEV
QLQQSGAELVKPGASVKMSCKASG
YTFTSYNMIHWVKQTPGQGLEWIGA
IYPGNGDTSYNQKFKGKATLTADKS
SSTAYMQLSSLTSEDSADYYCARSN
YYGSSWFFDVWGAGTTVTVSS
38 DIVLTQSPAILSASPGEKVTMTCRAS Anti-CD20 Leu16 scFv
SSVNYMDWYQKKPGSSPKPWIYAT light chain variable
SNLASGVPARFSGSGSGTSYSLTISR region
VEAEDAATYYCQQWSFNPPTFGGG
TKLEIK
39 RASSSVNYMD Anti-CD20 Leu16
scFv
light chain CDR1
40 ATSNLAS Anti-CD20 Leul 6
scFv
light chain CDR2
41 QQWSFNPPT Anti-CD20 Leu16
scFv
light chain CDR3
42 EVQLQQSGAELVKPGASVKMSCKA Anti-CD20 Leul6 scFv
SGYTFTSYNMEIWVKQTPGQGLEWI heavy chain
GAIYPGNGDTSYNQKFKGKATLTA
DKSSSTAYMQLSSLTSEDSADYYCA
RSNYYGSSWFFDVWGAGTTVTVS
43 SYNN4H Anti-CD20 Leul 6
scFv
heavy chain CDR1
44 AIYPGNGDTSYNQKFKG Anti-CD20 Leul6
scFv
heavy chain CDR2
1005931 In some embodiments, the hinge domain of the CD20 CAR comprises a CD8a
hinge
domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a
hinge
163
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
domain comprises or consists of an amino acid sequence set forth in SEQ ID
NO:9 or an amino
acid sequence that is at least 80% identical (e.g., at least 80%, at least
85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the amino acid
sequence set forth in SEQ ID NO:9. In some embodiments, the hinge domain
comprises a CD28
hinge domain, for example, a human CD28 hinge domain. In some embodiments, the
CD28
hinge domain comprises or consists of an amino acid sequence set forth in SEQ
ID NO:10 or an
amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the
amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the hinge
domain
comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In
some
embodiments, the IgG4 hinge domain comprises or consists of an amino acid
sequence set forth
in SEQ ID NO:11 or SEQ ID NO:12, or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO: 11 or
SEQ ID NO:12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-
Ch3
domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments,
the IgG4
hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:13 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:13.
1005941 In some embodiments, the transmembrane domain of the CD20 CAR
comprises a
CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In
some
embodiments, the CD8a transmembrane domain comprises or consists of an amino
acid
sequence set forth in SEQ ID NO:14 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:14. In
some embodiments, the transmembrane domain comprises a CD28 transmembrane
domain, for
example, a human CD28 transmembrane domain. In some embodiments, the CD28
transmembrane domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO: 15 or an amino acid sequence that is at least 80% identical (e.g., at
least 80%, at least 85%,
164
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:15.
1005951 In some embodiments, the intracellular costimulatory domain of the
CD20 CAR
comprises a 4-1BB costimulatory domain, for example, a human 4-1BB
costimulatory domain.
In some embodiments, the 4-1BB costimulatory domain comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:16 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:16. In
some embodiments, the intracellular costimulatory domain comprises a CD28
costimulatory
domain, for example, a human CD28 costimulatory domain. In some embodiments,
the CD28
costimulatory domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:17 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:17.
1005961 In some embodiments, the intracellular signaling domain of the CD20
CAR comprises
a CD3 zeta (C) signaling domain, for example, a human CD3C signaling domain.
In some
embodiments, the CD3C signaling domain comprises or consists of an amino acid
sequence set
forth in SEQ ID NO:18 or an amino acid sequence that is at least 80% identical
(e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:18.
1005971 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
comprising the CD20-specific scEv having sequences set forth in SEQ ID NO.37,
the CD8a
hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO:14,
the 4-1BB
costimulatory domain of SEQ ID NO: 16, the CD3C- signaling domain of SEQ ID
NO:18, and/or
variants (i.e., having a sequence that is at least 80% identical, for example,
at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99 identical
to the disclosed sequence) thereof.
1005981 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37,
the CD28
165
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
hinge domain of SEQ ID NO: O, the CD8a transmembrane domain of SEQ ID NO: IA,
the 4-
1BB costimulatory domain of SEQ ID NO:16, the CD3 C signaling domain of SEQ ID
NO:18,
and/or variants (i.e., having a sequence that is at least 80% identical, for
example, at least 80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99
identical to the disclosed sequence) thereof.
1005991 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37,
the IgG4 hinge
domain of SEQ ID NO:11 or SEQ ID NO:12, the CD8a transmembrane domain of SEQ
ID
NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
1006001 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
comprising the CD20-specific scFy having sequences set forth in SEQ ID NO:37,
the CD8a
hinge domain of SEQ ID NO:9, the CD28 transmembrane domain of SEQ ID NO:15,
the 4-1BB
costimulatory domain of SEQ ID NO:16, the CD3 C signaling domain of SEQ ID
NO:18, and/or
variants (i.e., having a sequence that is at least 80% identical, for example,
at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99 identical
to the disclosed sequence) thereof.
1006011 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
comprising the CD20-specific scFy having sequences set forth in SEQ ID NO:37,
the CD28
hinge domain of SEQ ID NO:10, the CD28 transmembrane domain of SEQ ID NO:15,
the 4-
1BB costimulatory domain of SEQ ID NO:16, the CD3 signaling domain of SEQ ID
NO:18,
and/or variants (i.e., having a sequence that is at least 80% identical, for
example, at least 80%,
at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99
identical to the disclosed sequence) thereof.
1006021 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD20 CAR, including, for example, a
CD20 CAR
166
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
comprising the CD20-specific scFv having sequences set forth in SEQ ID NO:37,
the IgG4 hinge
domain of SEQ ID NO:11 or SEQ ID NO:1, the CD28 transmembrane domain of SEQ ID

NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
CD22 CAR
1006031 In some embodiments, the CAR is a CD22 CAR ("CD22-CAR"), and in these
embodiments, the polycistronic vector comprises an expression cassette that
contains a
nucleotide sequence encoding a CD22 CAR. CD22, which is a transmembrane
protein found
mostly on the surface of mature B cells that functions as an inhibitory
receptor for B cell receptor
(BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias
(e.g., B-
chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia
(ALL), and
Burkitt's lymphoma) and is not present on the cell surface in early stages of
B cell development
or on stem cells. In some embodiments, the CD22 CAR may comprise a signal
peptide, an
extracellular binding domain that specifically binds CD22, a hinge domain, a
transmembrane
domain, an intracellular costimulatory domain, and/or an intracellular
signaling domain in
tandem.
1006041 In some embodiments, the signal peptide of the CD22 CAR comprises a
CD8a signal
peptide. In some embodiments, the CD8a signal peptide comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:6 or an amino acid sequence that is at least
80% identical (e.g.,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO:6. In some
embodiments, the signal peptide comprises an Ig,K signal peptide. In some
embodiments, the
IgK signal peptide comprises or consists of an amino acid sequence set forth
in SEQ ID NO:7 or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the
signal peptide
comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-
a or
CSF2RA signal peptide comprises or consists of an amino acid sequence set
forth in SEQ ID
NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%, at
167
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% identical)
to the amino acid sequence set forth in SEQ ID NO:.
1006051 In some embodiments, the extracellular binding domain of the CD22 CAR
is specific to
CD22, for example, human CD22. The extracellular binding domain of the CD22
CAR can be
codon-optimized for expression in a host cell or to have variant sequences to
increase functions
of the extracellular binding domain. In some embodiments, the extracellular
binding domain
comprises an immunogenically active portion of an immunoglobulin molecule, for
example, an
scFv.
1006061 In some embodiments, the extracellular binding domain of the CD22 CAR
is derived
from an antibody specific to CD22, including, for example, SM03, inotuzumab,
epratuzumab,
moxetumomab, and pinatuzumab. In any of these embodiments, the extracellular
binding
domain of the CD22 CAR can comprise or consist of the VH, the VL, and/or one
or more CDRs
of any of the antibodies.
1006071 In some embodiments, the extracellular binding domain of the CD22 CAR
comprises
an scFv derived from the m971 monoclonal antibody (m971), which comprises the
heavy chain
variable region (VH) and the light chain variable region (VL) of m971
connected by a linker. In
some embodiments, the linker is a 3xG4S linker. In other embodiments, the
Whitlow linker may
be used instead. In some embodiments, the amino acid sequences of the entire
m971-derived
scFv (also referred to as m971 scFv) and its different portions are provided
in Table 16 below.
In some embodiments, the CD22-specific scFv comprises or consists of an amino
acid sequence
set forth in SEQ ID NO:45, 46, or 50, or an amino acid sequence that is at
least 80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO.45, 46,
or 50. In some embodiments, the CD22-specific scFv may comprise one or more
CDRs having
amino acid sequences set forth in SEQ ID NOs: 47-49 and 51-53. In some
embodiments, the
CD22-specific scFv may comprise a heavy chain with one or more CDRs having
amino acid
sequences set forth in SEQ ID NOs: 47-49. In some embodiments, the CD22-
specific scFv may
comprise a light chain with one or more CDRs having amino acid sequences set
forth in SEQ ID
NOs: 51-53. In any of these embodiments, the CD22-specific scFv may comprise
one or more
CDRs comprising one or more amino acid substitutions, or comprising a sequence
that is at least
80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%,
at least 96%, at least
168
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
97%, at least 98%, at least 99%, or 100% identical), to any of the sequences
identified. In some
embodiments, the extracellular binding domain of the CD22 CAR comprises or
consists of the
one or more CDRs as described herein.
1006081 In some embodiments, the extracellular binding domain of the CD22 CAR
comprises
an scFv derived from m971-L7, which is an affinity matured variant of m971
with significantly
improved CD22 binding affinity compared to the parental antibody m971
(improved from about
2 nM to less than 50 pM). In some embodiments, the scFv derived from m971-L7
comprises the
VH and the VL of m971-L7 connected by a 3xG4S linker. In other embodiments,
the Whitlow
linker may be used instead. In some embodiments, the amino acid sequences of
the entire m971-
L7-derived scFv (also referred to as m971-L7 scFv) and its different portions
are provided in
Table 16 below. In some embodiments, the CD22-specific scFv comprises or
consists of an
amino acid sequence set forth in SEQ ID NO:54, 55, or 59, or an amino acid
sequence that is at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid
sequence set forth in
SEQ ID NO:54, 55, or 59. In some embodiments, the CD22-specific scFv may
comprise one or
more CDRs having amino acid sequences set forth in SEQ ID NOs: 56-58 and 60-
62. In some
embodiments, the CD22-specific scFv may comprise a heavy chain with one or
more CDRs
having amino acid sequences set forth in SEQ ID NOs: 56-58. In some
embodiments, the CD22-
specific scFv may comprise a light chain with one or more CDRs having amino
acid sequences
set forth in SEQ ID NOs: 60-62. In any of these embodiments, the CD22-specific
scFv may
comprise one or more CDRs comprising one or more amino acid substitutions, or
comprising a
sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at
least 90%, at least 95%,
at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to
any of the sequences
identified. In some embodiments, the extracellular binding domain of the CD22
CAR comprises
or consists of the one or more CDRs as described herein.
Table 16. Exemplary sequences of anti-CD22 scFv and components
SEQ ID NO: Amino Acid Sequence Description
45 QVQLQQSGPGLVKPSQTLSLTCAISG Anti-CD22 m971 scFv
DSVSSNSAAWNWIRQSPSRGLEWL entire sequence, with
GRTYYRSKWYNDYAVSVKSRITINP 3xG4S linker
DTSKNQFSLQLNSVTPEDTAVYYCA
REVTGDLEDAFDIWGQGTMVTVSS
169
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
GGGGSGGGGSGGGGSDIQMTQSPSS
LSASVGDRVTITCRASQTIWSYLNW
YQQRPGKAPNLLIYAASSLQSGVPS
RFSGRGSGTDFTLTISSLQAEDFATY
YCQQSYSIPQTFGQGTKLEIK
46 QVQLQQSGPGLVKPSQTLSLTCAISG Anti-CD22 m971 scFv
DSVSSNSAAWNWIRQSPSRGLEWL heavy chain variable
GRTYYRSKWYNDYAVSVKSRITINP region
DTSKNQFSLQLNSVTPEDTAVYYCA
REVTGDLEDAFDIWGQGTMVTVSS
47 GDSVSSNSAA Anti-CD22 m971
scFv
heavy chain CDR1
48 TYYRSKWYN Anti-CD22 m971
scFv
heavy chain CDR2
49 AREVTGDLEDAFDI Anti-CD22 m971
scFv
heavy chain CDR3
50 DIQMTQSPSSLSASVGDRVTITCRAS Anti-CD22 m971 scFv
QTIWSYLNWYQQRPGKAPNLLIYA light chain
ASSLQSGVPSRFSGRGSGTDFTLTISS
LQAEDFATYYCQQSYSIPQTFGQGT
KLEIK
51 QTIWSY Anti-CD22 m971
scFv
light chain CDR1
52 AAS Anti-CD22 m971
scFv
light chain CDR2
53 QQSYSIPQT Anti-CD22 m971
scFv
light chain CDR3
54 QVQLQQSGPGMVKPSQTLSLTCAIS Anti-CD22 m971-L7
GDSVSSNSVAWNWIRQSPSRGLEW scFv entire sequence,
LGRTYYRSTWYNDYAVSMKSRITIN with 3xG4S linker
PDTNKNQFSLQLNSVTPEDTAVYYC
AREVTGDLEDAFDIWGQGTMVTVS
SGGGGSGGGGSGGGGSDIQMIQSPS
SLSASVGDRVTITCRASQTIWSYLN
WYRQRPGEAPNLLIYAASSLQSGVP
SRFSGRGSGTDFTLTISSLQAEDFAT
YYCQQSYSIPQTFGQGTKLEIK
170
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
55 QVQLQQSGPGMVKPSQTLSLTCAIS Anti-CD22 m971-L7
GDSVSSNSVAWNWIRQSPSRGLEW scFv heavy chain
LGRTYYRSTWYNDYAVSMKSRITIN variable region
PDTNKNQFSLQLNSVTPEDTAVYYC
AREVTGDLEDAFDIWGQGTMVTVS
56 GDSVSSNSVA Anti-CD22 m971-L7
scFv heavy chain CDR1
57 TYYRSTWYN Anti-CD22 m971-L7
scFv heavy chain CDR2
58 AREVTGDLEDAFDI Anti-CD22 m971-L7
scFv heavy chain CDR3
59 DIQMIQSPS SL S A SVGDRVTITCRA S Anti-CD22 m971-L7
QTIWSYLNWYRQRPGEAPNLLIYAA scFv light chain variable
SSLQSGVPSRFSGRGSGTDFTLTISSL region
QAEDFATYYCQQSYSIPQTFGQGTK
LEIK
60 QTIWSY Anti-CD22 m971-L7
scFv light chain CDR1
61 AAS Anti-CD22 m971-L7
scFv light chain CDR2
62 QQSYSIPQT Anti-CD22 m971-L7
scFv light chain CDR3
1006091 In some embodiments, the extracellular binding domain of the CD22 CAR
comprises
immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents
that
comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can
bind to the surface of
the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a
dsFy of an anti-
CD22 antibody, RFB4, fused to a 38-1(Da truncated form of Pseudomonas exotoxin
A (Bang et
al., Clin. Cancer Res., 11:1545-50 (2005)). HA22 (CAT8015, moxetumomab
pasudotox) is a
mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1):
607-17 (2005)).
Suitable sequences of antigen binding domains of HA22 and BL22 specific to
CD22 are
disclosed in, for example, U.S. Patent Nos. 7,541,034; 7,355,012; and
7,982,011, which are
hereby incorporated by reference in their entirety.
1006101 In some embodiments, the hinge domain of the CD22 CAR comprises a CD8a
hinge
domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a
hinge
171
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
domain comprises or consists of an amino acid sequence set forth in SEQ ID
NO:9 or an amino
acid sequence that is at least 80% identical (e.g., at least 80%, at least
85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the amino acid
sequence set forth in SEQ ID NO:9. In some embodiments, the hinge domain
comprises a CD28
hinge domain, for example, a human CD28 hinge domain. In some embodiments, the
CD28
hinge domain comprises or consists of an amino acid sequence set forth in SEQ
ID NO:10 or an
amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the
amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the hinge
domain
comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In
some
embodiments, the IgG4 hinge domain comprises or consists of an amino acid
sequence set forth
in SEQ ID NO:11 or SEQ ID NO:12, or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO: 11 or
SEQ ID NO:12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-
Ch3
domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments,
the IgG4
hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:13 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:13.
1006111 In some embodiments, the transmembrane domain of the CD22 CAR
comprises a
CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In
some
embodiments, the CD8a transmembrane domain comprises or consists of an amino
acid
sequence set forth in SEQ ID NO:14 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:14. In
some embodiments, the transmembrane domain comprises a CD28 transmembrane
domain, for
example, a human CD28 transmembrane domain. In some embodiments, the CD28
transmembrane domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO: 15 or an amino acid sequence that is at least 80% identical (e.g., at
least 80%, at least 85%,
172
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:15.
1006121 In some embodiments, the intracellular costimulatory domain of the
CD22 CAR
comprises a 4-1BB costimulatory domain, for example, a human 4-1BB
costimulatory domain.
In some embodiments, the 4-1BB costimulatory domain comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:16 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:16. In
some embodiments, the intracellular costimulatory domain comprises a CD28
costimulatory
domain, for example, a human CD28 costimulatory domain. In some embodiments,
the CD28
costimulatory domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:17 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:17.
1006131 In some embodiments, the intracellular signaling domain of the CD22
CAR comprises
a CD3 zeta (C) signaling domain, for example, a human CD3C signaling domain.
In some
embodiments, the CD3C signaling domain comprises or consists of an amino acid
sequence set
forth in SEQ ID NO:18 or an amino acid sequence that is at least 80% identical
(e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:18.
1006141 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
comprising the CD22-specific scFv having sequences set forth in SEQ ID NO.45
or SEQ ID
NO:54, the CD8a hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of
SEQ ID
NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3C signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
1006151 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45
or SEQ ID
173
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NO:54, the CD28 hinge domain of SEQ ID NO:10, the CD8a transmembrane domain of
SEQ ID
NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 C signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
1006161 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45
or SEQ ID
NO:54, the IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD8a
transmembrane
domain of SEQ ID NO:14, the 4-1BB costimulatory domain of SEQ ID NO:16, the
CD3
signaling domain of SEQ ID NO:18, and/or variants (i.e., having a sequence
that is at least 80%
identical, for example, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least
97%, at least 98%, or at least 99 identical to the disclosed sequence)
thereof.
1006171 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
comprising the CD22-specific scFy having sequences set forth in SEQ ID NO:45
or SEQ ID
NO:54, the CD8a hinge domain of SEQ ID NO:9, the CD28 transmembrane domain of
SEQ ID
NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 C signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
1006181 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
comprising the CD22-specific scFy having sequences set forth in SEQ ID NO:45
or SEQ ID
NO:54, the CD28 hinge domain of SEQ ID NO:10, the CD28 transmembrane domain of
SEQ ID
NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the CD3 signaling
domain of SEQ
ID NO:18, and/or variants (i.e., having a sequence that is at least 80%
identical, for example, at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, or at
least 99 identical to the disclosed sequence) thereof.
1006191 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a CD22 CAR, including, for example, a
CD22 CAR
174
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
comprising the CD22-specific scFv having sequences set forth in SEQ ID NO:45
or SEQ ID
NO:54, the IgG4 hinge domain of SEQ ID NO:11 or SEQ ID NO:12, the CD28
transmembrane
domain of SEQ ID NO:15, the 4-1BB costimulatory domain of SEQ ID NO:16, the
CD3C
signaling domain of SEQ ID NO:18, and/or variants (i.e., having a sequence
that is at least 80%
identical, for example, at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at least
97%, at least 98%, or at least 99 identical to the disclosed sequence)
thereof.
BCMA CAR
1006201 In some embodiments, the CAR is a BCMA CAR ("BCMA-CAR"), and in these
embodiments, the polycistronic vector comprises an expression cassette that
contains a
nucleotide sequence encoding a BCMA CAR. BCMA is a tumor necrosis family
receptor
(TNFR) member expressed on cells of the B cell lineage, with the highest
expression on
terminally differentiated B cells or mature B lymphocytes. BCMA is involved in
mediating the
survival of plasma cells for maintaining long-term humoral immunity. The
expression of BCMA
has been recently linked to a number of cancers, such as multiple myeloma,
Hodgkin's and non-
Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments,
the BCMA
CAR may comprise a signal peptide, an extracellular binding domain that
specifically binds
BCMA, a hinge domain, a transmembrane domain, an intracellular costimulatory
domain, and/or
an intracellular signaling domain in tandem.
1006211 In some embodiments, the signal peptide of the BCMA CAR comprises a
CD8a signal
peptide. In some embodiments, the CD8a signal peptide comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:6 or an amino acid sequence that is at least
80% identical (e.g.,
at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID
NO:6. In some
embodiments, the signal peptide comprises an Ig,K signal peptide. In some
embodiments, the
IgK signal peptide comprises or consists of an amino acid sequence set forth
in SEQ ID NO:7 or
an amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least
90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or
100% identical) to
the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the
signal peptide
comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-
a or
CSF2RA signal peptide comprises or consists of an amino acid sequence set
forth in SEQ ID
NO:8 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%, at
175
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100% identical)
to the amino acid sequence set forth in SEQ ID NO:.
1006221 In some embodiments, the extracellular binding domain of the BCMA CAR
is specific
to BCMA, for example, human BCMA. The extracellular binding domain of the BCMA
CAR
can be codon-optimized for expression in a host cell or to have variant
sequences to increase
functions of the extracellular binding domain.
1006231 In some embodiments, the extracellular binding domain comprises an
immunogenically
active portion of an immunoglobulin molecule, for example, an scFv. In some
embodiments, the
extracellular binding domain of the BCMA CAR is derived from an antibody
specific to BCMA,
including, for example, belantamab, erlanatamab, teclistamab, LCAR-B38M, and
ciltacabtagene.
In any of these embodiments, the extracellular binding domain of the BCMA CAR
can comprise
or consist of the VH, the VL, and/or one or more CDRs of any of the
antibodies.
1006241 In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
an scFv derived from Cl 1D5.3, a murine monoclonal antibody as described in
Carpenter et al.,
Clin. Cancer Res. 19(8):2048-2060 (2013). See also PCT Application Publication
No.
W02010/104949. The C11D5.3-derived scFv may comprise the heavy chain variable
region
(VH) and the light chain variable region (VL) of Cl 1D5.3 connected by the
Whitlow linker, the
amino acid sequences of which is provided in Table 17 below. In some
embodiments, the
BCMA-specific extracellular binding domain comprises or consists of an amino
acid sequence
set forth in SEQ ID NO:63, 64, or 68, or an amino acid sequence that is at
least 80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:63, 64,
or 68. In some embodiments, the BCMA-specific extracellular binding domain may
comprise
one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-67
and 69-71. In
some embodiments, the BCMA-specific extracellular binding domain may comprise
a light chain
with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 65-
67. In some
embodiments, the BCMA-specific extracellular binding domain may comprise a
heavy chain
with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 69-
71. In any of
these embodiments, the BCMA-specific scFv may comprise one or more CDRs
comprising one
or more amino acid substitutions, or comprising a sequence that is at least
80% identical (e.g., at
least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
97%, at least 98%, at
176
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
least 99%, or 100% identical), to any of the sequences identified. In some
embodiments, the
extracellular binding domain of the BCMA CAR comprises or consists of the one
or more CDRs
as described herein.
1006251 In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
an scFv derived from another murine monoclonal antibody, Cl2A3 2, as described
in Carpenter
et al., Clin. Cancer Res. 19(8):2048-2060 (2013) and PCT Application
Publication No.
W02010/104949, the amino acid sequence of which is also provided in Table 17
below. In
some embodiments, the BCMA-specific extracellular binding domain comprises or
consists of an
amino acid sequence set forth in SEQ ID NO:72, 73, or 77, or an amino acid
sequence that is at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid
sequence set forth in
SEQ ID NO:72, 73, or 77. In some embodiments, the BCMA-specific extracellular
binding
domain may comprise one or more CDRs having amino acid sequences set forth in
SEQ ID NOs:
74-76 and 78-80. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise a light chain with one or more CDRs having amino acid sequences set
forth in SEQ ID
NOs: 74-76. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise a heavy chain with one or more CDRs having amino acid sequences set
forth in SEQ
ID NOs: 78-80. In any of these embodiments, the BCMA-specific scFv may
comprise one or
more CDRs comprising one or more amino acid substitutions, or comprising a
sequence that is at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical), to any of the
sequences identified. In
some embodiments, the extracellular binding domain of the BCMA CAR comprises
or consists
of the one or more CDRs as described herein.
1006261 In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
a murine monoclonal antibody with high specificity to human BCMA, referred to
as BB2121 in
Friedman et al., Hum. Gene Ther. 29(5):585-601 (2018)). See also, PCT
Application Publication
No. W02012163805.
1006271 In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
single variable fragments of two heavy chains (VHH) that can bind to two
epitopes of BCMA as
described in Zhao et al., J. Hematol. Oncol. 11(1):141 (2018), also referred
to as LCAR-B38M.
See also, PCT Application Publication No. W02018/028647.
177
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00628] In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
a fully human heavy-chain variable domain (FHVH) as described in Lam et al.,
Nat. Commun.
11(1):283 (2020), also referred to as FHVH33. See also, PCT Application
Publication No.
W02019/006072. The amino acid sequences of FHVH33 and its CDRs are provided in
Table
17 below. In some embodiments, the BCMA-specific extracellular binding domain
comprises or
consists of an amino acid sequence set forth in SEQ ID NO:81 or an amino acid
sequence that is
at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid
sequence set forth in
SEQ ID NO:81. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs:
82-84. In
any of these embodiments, the BCMA-specific extracellular binding domain may
comprise one
or more CDRs comprising one or more amino acid substitutions, or comprising a
sequence that is
at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at
least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical), to any of the
sequences identified. In
some embodiments, the extracellular binding domain of the BCMA CAR comprises
or consists
of the one or more CDRs as described herein.
[00629] In some embodiments, the extracellular binding domain of the BCMA CAR
comprises
an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No.
11,026,975 B2, the
amino acid sequence of which is provided in Table 17 below. In some
embodiments, the
BCMA-specific extracellular binding domain comprises or consists of an amino
acid sequence
set forth in SEQ ID NO: 118, 119, or 123, or an amino acid sequence that is at
least 80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO: 118,
119, or 123. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise one or more CDRs having amino acid sequences set forth in SEQ ID NOs:
120-122
and 124-126. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise a light chain with one or more CDRs having amino acid sequences set
forth in SEQ ID
NOs: 120-122. In some embodiments, the BCMA-specific extracellular binding
domain may
comprise a heavy chain with one or more CDRs having amino acid sequences set
forth in SEQ
ID NOs: 124-126. In any of these embodiments, the BCMA-specific scFv may
comprise one or
more CDRs comprising one or more amino acid substitutions, or comprising a
sequence that is at
178
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical), to any of the
sequences identified. In
some embodiments, the extracellular binding domain of the BCMA CAR comprises
or consists
of the one or more CDRs as described herein.
1006301 Additionally, CARs and binders directed to BCMA have been described in
U.S.
Application Publication Nos. 2020/0246381 Al and 2020/0339699 Al, the entire
contents of
each of which are incorporated by reference herein.
Table 17. Exemplary sequences of anti-BCMA binder and components
SEQ ID NO: Amino Acid Sequence Description
63 DIVLTQSPASLAMSLGKRATISCRAS Anti-BCMA Cl 1D5.3
ESVSVIGAHLIHWYQQKPGQPPKLLI scFv entire sequence,
YLASNLETGVPARFSGSGSGTDFTLT with Whitlow linker
IDPVEEDDVAIYSCLQSRIFPRTFGG
GTKLEIKGSTSGSGKPGSGEGSTKG
QIQLVQSGPELKKPGETVKISCKASG
YTFTDYSINWVKRAPGKGLKWMG
WINTETREPAYAYDFRGRFAFSLETS
ASTAYLQINNLKYEDTATYFCALDY
SYAMDYWGQGTSVTVSS
64 DIVLTQSPASLAMSLGKRATISCRAS Anti-BCMA Cl 1D5.3
ESVSVIGAHLIFIWYQQKPGQPPKLLI scFv light chain variable
YLASNLETGVPARFSGSGSGTDFTLT region
IDPVEEDDVAIYSCLQSRIFPRTFGG
GTKLEIK
65 RASESVSVIGAHLIH Anti-BCMA Cl
1D5.3
scFv light chain CDR1
66 LASNLET Anti-BCMA Cl
1D5.3
scFv light chain CDR2
67 LQSRIFPRT Anti-BCMA Cl
1D5.3
scFv light chain CDR3
68 QIQLVQSGPELKKPGETVKISCKASG Anti-BCMA Cl 1D5.3
YTFTDYSINWVKRAPGKGLKWMG scFv heavy chain
WINTETREPAYAYDFRGRFAFSLETS variable region
ASTAYLQINNLKYEDTATYFCALDY
SYAMDYWGQGTSVTVSS
69 DYSIN Anti-BCMA Cl
1D5.3
scFv heavy chain CDR1
179
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
70 WINTETREPAYAYDFRG Anti-BCMA Cl
1D5.3
scFv heavy chain CDR2
71 DYSYAMDY Anti-BCMA Cl
1D5.3
scFv heavy chain CDR3
72 DIVLTQSPPSLAMSLGKRATISCRAS Anti-BCMA Cl2A3.2
ESVTILGSHLIYWYQQKPGQPPTLLI scFv entire sequence,
QLASNVQTGVPARFSGSGSRTDFTL with Whitlow linker
TIDPVEEDDVAVYYCLQSRTIPRTFG
GGTKLEIKGSTSGSGKPGSGEGSTK
GQIQLVQSGPELKKPGETVKISCKAS
GYTFRHYSMNWVKQAPGKGLKWM
GRINTESGVPIYADDFKGRFAFSVET
SASTAYLVINNLKDEDTASYFCSND
YLYSLDFWGQGTALTVSS
73 DIVLTQ SPP SLAM SLGKRATISCRAS Anti-BCMA Cl2A3.2
ESVTILGSHLIYWYQQKPGQPPTLLI scFv light chain variable
QLASNVQTGVPARFSGSGSRTDFTL region
TIDPVEEDDVAVYYCLQSRTIPRTFG
GGTKLEIK
74 RASESVTILGSHLIY Anti-BCMA C
12A3.2
scFv light chain CDR1
75 LASNVQT Anti-BCMA C12A3 2
scFv light chain CDR2
76 LQSRTIPRT Anti-BCMA C12A3.2
scFv light chain CDR3
77 QIQLVQSGPELKKPGETVKISCKASG Anti-BCMA C 12A3.2
YTFRHYSMNWVKQAPGKGLKWMG scFv heavy chain
RINTESGVPIYADDFKGRFAFSVETS variable region
ASTAYLVINNLKDEDTASYFCSNDY
LYSLDFWGQGTALTVSS
78 HYSMN Anti-BCMA C12A3.2
scFv heavy chain CDR1
79 RINTESGVPIYADDFKG Anti-BCMA C12A3.2
scFv heavy chain CDR2
80 DYLYSLDF Anti-BCMA C12A3.2
scFv heavy chain CDR3
81 EVQLLESGGGLVQPGGSLRLSCAAS Anti-BCMA FHVH33
GFTFSSYAMSWVRQAPGKGLEWVS entire sequence
SISGSGDYIYYADSVKGRFTISRDISK
180
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
NTLYLQMNSLRAEDTAVYYCAKEG
TGANSSLADYRGQGTLVTVSS
82 GFTFSSYA Anti-BCMA FHVH33
CDR1
83 ISGSGDYI Anti-BCMA FHVH33
CDR2
84 AKEGTGANSSLADY Anti-BCMA FHVH33
CDR3
118 DIQMTQSPSSLSASVGDRVTITCRAS Anti-BCMA CT103A
QSISSYLNWYQQKPGKAPKLLIYAA scFv entire sequence,
SSLQSGVPSRFSGSGSGTDFTLTISSL with Whitlow linker
QPEDFATYYCQQKYDLLTFGGGTK
VEIKGSTSGSGKPGSGEGSTKGQLQ
LQESGPGLVKPSETLSLTCTVSGGSI
SSSSYYWGWIRQPPGKGLEWIGSISY
SGSTYYNPSLKSRVTISVDTSKNQF S
LKLSSVTAADTAVYYCARDRGDTIL
DVWGQGTMVTVSS
119 DIQMTQSPSSLSASVGDRVTITCRAS Anti-BCMA CT103A
QSISSYLNWYQQKPGKAPKLLIYAA scFv light chain variable
SSLQSGVPSRFSGSGSGTDFTLTISSL region
QPEDFATYYCQQKYDLLTFGGGTK
VEIK
120 QSISSY Anti-BCMA CT103A
scFv light chain CDR1
121 AAS Anti-BCMA CT103A
scFv light chain CDR2
122 QQKYDLLT Anti-BCMA CT103A
scFv light chain CDR3
123 QLQLQESGPGLVKPSETLSLTCTVSG Anti-BCMA CT103A
GSISSSSYYWGWIRQPPGKGLEWIGS scFv heavy chain
ISYSGSTYYNPSLKSRVTISVDTSKN variable region
QFSLKLSSVTAADTAVYYCARDRG
DT1LDVWGQGTMVTVSS
124 GGSISSSSYY Anti-BCMA CT103A
scFv heavy chain CDR1
125 ISYSGST Anti-BCMA CT103A
scFv heavy chain CDR2
181
CA 03225283 2024- 1- 8

WO 2023/287827 PCT/US2022/036874
SEQ ID NO: Amino Acid Sequence Description
126 ARDRGDTILDV Anti-BCMA CT103A
scFy heavy chain CDR3
1006311 In some embodiments, the hinge domain of the BCMA CAR comprises a CD8a
hinge
domain, for example, a human CD8a hinge domain. In some embodiments, the CD8a
hinge
domain comprises or consists of an amino acid sequence set forth in SEQ ID
NO:9 or an amino
acid sequence that is at least 80% identical (e.g., at least 80%, at least
85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the amino acid
sequence set forth in SEQ ID NO:9. In some embodiments, the hinge domain
comprises a CD28
hinge domain, for example, a human CD28 hinge domain. In some embodiments, the
CD28
hinge domain comprises or consists of an amino acid sequence set forth in SEQ
ID NO: 10 or an
amino acid sequence that is at least 80% identical (e.g., at least 80%, at
least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%
identical) to the
amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the hinge
domain
comprises an IgG4 hinge domain, for example, a human IgG4 hinge domain. In
some
embodiments, the IgG4 hinge domain comprises or consists of an amino acid
sequence set forth
in SEQ ID NO: ii or SEQ ID NO:12, or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:11 or
SEQ ID NO:12. In some embodiments, the hinge domain comprises a IgG4 hinge-Ch2-
Ch3
domain, for example, a human IgG4 hinge-Ch2-Ch3 domain. In some embodiments,
the IgG4
hinge-Ch2-Ch3 domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:13 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:13.
1006321 In some embodiments, the transmembrane domain of the BCMA CAR
comprises a
CD8a transmembrane domain, for example, a human CD8a transmembrane domain. In
some
embodiments, the CD8a transmembrane domain comprises or consists of an amino
acid
sequence set forth in SEQ ID NO:14 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO: 14. In
182
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some embodiments, the transmembrane domain comprises a CD28 transmembrane
domain, for
example, a human CD28 transmembrane domain. In some embodiments, the CD28
transmembrane domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:15 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:15.
1006331 In some embodiments, the intracellular costimulatory domain of the
BCMA CAR
comprises a 4-1BB costimulatory domain, for example, a human 4-1BB
costimulatory domain.
In some embodiments, the 4-1BB costimulatory domain comprises or consists of
an amino acid
sequence set forth in SEQ ID NO:16 or an amino acid sequence that is at least
80% identical
(e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,
at least 97%, at least
98%, at least 99%, or 100% identical) to the amino acid sequence set forth in
SEQ ID NO:16. In
some embodiments, the intracellular costimulatory domain comprises a CD28
costimulatory
domain, for example, a human CD28 costimulatory domain. In some embodiments,
the CD28
costimulatory domain comprises or consists of an amino acid sequence set forth
in SEQ ID
NO:17 or an amino acid sequence that is at least 80% identical (e.g., at least
80%, at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:17.
1006341 In some embodiments, the intracellular signaling domain of the BCMA
CAR comprises
a CD3 zeta () signaling domain, for example, a human CD3t signaling domain. In
some
embodiments, the CD3 C signaling domain comprises or consists of an amino acid
sequence set
forth in SEQ ID NO:18 or an amino acid sequence that is at least 80% identical
(e.g., at least
80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at
least 98%, at least
99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO:18.
1006351 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a BCMA CAR, including, for example, a
BCMA CAR
comprising any of the BCMA-specific extracellular binding domains as
described, the CD8a
hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO:14,
the 4-1BB
costimulatory domain of SEQ ID NO: 16, the CD3 signaling domain of SEQ ID
NO:18, and/or
variants (i.e., having a sequence that is at least 80% identical, for example,
at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99 identical
183
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR
may
additionally comprise a signal peptide (e.g., a CD8a signal peptide) as
described.
1006361 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a BCMA CAR, including, for example, a
BCMA CAR
comprising any of the BCMA-specific extracellular binding domains as
described, the CD8a
hinge domain of SEQ ID NO:9, the CD8a transmembrane domain of SEQ ID NO:14,
the CD28
costimulatory domain of SEQ ID NO 17, the CD3C, signaling domain of SEQ ID NO
18, and/or
variants (i.e., having a sequence that is at least 80% identical, for example,
at least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or
at least 99 identical
to the disclosed sequence) thereof. In any of these embodiments, the BCMA CAR
may
additionally comprise a signal peptide as described.
1006371 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a BCMA CAR as set forth in SEQ ID
NO:127 or is at
least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least
95%, at least 96%, at
least 97%, at least 98%, at least 99%, or 100% identical) to the nucleotide
sequence set forth in
SEQ ID NO:127 (see Table 18). The encoded BCMA CAR has a corresponding amino
acid
sequence set forth in SEQ ID NO:128 or is at least 80% identical (e.g., at
least 80%, at least
85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%
identical) to the amino acid sequence set forth in SEQ ID NO:128, with the
following
components: CD8a signal peptide, CT103A scFv (VL-Whitlow linker-VH), CD8a
hinge domain,
CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3C signaling
domain.
1006381 In some embodiments, the polycistronic vector comprises an expression
cassette that
contains a nucleotide sequence encoding a commercially available embodiment of
BCMA CAR,
including, for example, idecabtagene vicleucel (ide-cel, also called bb2121).
In some
embodiments, the polycistronic vector comprises an expression cassette that
contains a
nucleotide sequence encoding idecabtagene vicleucel or portions thereof.
Idecabtagene vicleucel
comprises a BCMA CAR with the following components: the BB2121 binder, CD8a
hinge
domain, CD8a transmembrane domain, 4-1BB costimulatory domain, and CD3
signaling
domain.
184
CA 03225283 2024- 1- 8

9 -1 -17Z0Z E9ZSZZ0
SS I
VICEVVIASSIXIS dONNSICEASLIAISNIS
dNIA AISOS A SISDIMTION9ddOXIMDMA
AS S SSISDOSAIDEISIIHS d)IAI-DdDSHOI
6169)1ISOJOS9d)I0S9SISONTIANIDO
dITICIANOODAAIV.ICEHdOIS
aouanbas SOSOS 411SdADSOIS SVVAIII)IdV)I9d)I
PPE cmIum 11VD OOAMNIAS SISOSVIIDILLAIRDASVSISS
VIAIDEE A-IBIdumxa 8Z I
au000000ET000EguoETuouogT000FauFau
ToovauSSRpoovooSoauoSalooSSSuoauiSlooSSIa
ouooE5guuoFEoFoORuFFoffuEoEguaTuoEFoiuffu
5oguaulooggaoo55Taucauguuuguotouu5ounul
SpoRSE-eSS-e00000ueRueSSouRe000RuuuFRoFRETu
Re5000au5E5oo5Rugu55o5Ruau55io515oaanTguE
uuSiSSouguaSISSToogutoSaoueauTtoSuo0EESEOE
gFgeogeoovTooFT0000RougoogooTegeogeoTTFueFTS'
ugu5TouugT_Tuaugftauu5uauuftooTnaooTo
ETFToFFIeFRuS'ffuffevoTopTovvvouTffuoopffuSiumpoo
uuoRnoTTuTuTuTTooTouu'u5Ru'ugeo5auuuoRao
auoou'uotomtooauoTuSTSSTooSaTototoSTSoSS
12Toouo0ooToT0000ggToTuouToTuouogToogono
uSETooEFatoououoFTEooguSEoESooFToEioauguTE
loogua000,SgoglopTSTolooguooguoogoTRuouu0000
Reopooe000Too-auT00005T000moo-poouToo-eueo353
ooFloonFTS000FigoneopauolSoauoiFFInuouTFFF.0
olESSETuiSouRuiouwoarauSESSiSoTuguStooSoSTo
mouiFiggogFouougEoFooFooaigiongugiogualoo
NongeaoueOucooi0o-cougeTOoomEoouolgeOaTge0
uuoT000lF000ueouiouToouo0205012-uTuTooToiuTStOF
T_Tai.gaTo'55uu5gu00000f'u0000l.'aTo
FRTomounffuTFm.ffuoStopooToFFTSSToloTFTouoFToo
uoi000l2Tooaugaonooguioage000Di2
aSuoSToguo5ToSuouSSSuuuouoguoSSEaoSSTolo
WToogueoSgoologoguoauoguogauRuol-ugang&
uoauFEauF5oFEnnouoTooToauFaumuuuoRuoTETauT
aunaguotTnauutooRuotolguoffuowoauoloTauon
TeReouF5R4oTeSSTR-eoR512-eon5gueoTuooDTS5F5TRu
RuoFmgrooTeoFToFluTop5TooTogum0000StRuFFRu
oauuuguoguoTuTgRneRumuloguogunuogEgEolgRuo
aouanb as gFgoognouoieoaeoTgauouguggeTFToieoEToTgT000
apnbaionu 1-v3 loowooToTgu000aTuguooTeoaooftoo5000uool.
vyug ktuiclulaxa otoSn.005OpFoo5Toop5n.00FooutgeoompoSSIe LZ I
U091:1!.13SJa J3uanbas :ON CU Oas
slivp ywpg jo saouanbas Kruidulaxa .sj am",
tL,890/ZZOZSf1/Id L,Z8L,8Z/2OZ OAA

WO 2023/287827
PCT/US2022/036874
SEQ ID NO: Sequence
Description
VYYCARDRGDTILDVWGQGTMVTVSSFV
PVFLPAKPTTTPAPRPPTPAPTIASQPLSLR
PEACRPAAGGAVHTRGLDFACDIYIWAPL
AGTCGVLLLSLVITLYCNHRNKRGRKKLL
YIFKQPFMRPVQTTQEEDGCSCRFPEEEEG
GCELRVKFSRSADAPAYQQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRK
NPQEGLYNELQKDKMAEAYSEIGMKGER
RRGKGHDGLYQGLSTATKDTYDALHMQ
ALPPR
R. Characteristics of Hypoimmunogenic Cells
1006391 In some embodiments, the population of hypoimmunogenic stem cells
retains
pluripotency as compared to a control stem cell (e.g., a wild-type stem cell
or immunogenic stem
cell). In some embodiments, the population of hypoimmunogenic stem cells
retains
differentiation potential as compared to a control stem cell (e.g., a wild-
type stem cell or
immunogenic stem cell).
1006401 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
immune activation in the subject or patient. In some instances, the level of
immune activation
elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%
lower compared to the level of immune activation produced by the
administration of
immunogenic cells. In some embodiments, the administered population of
hypoimmunogenic
cells fails to elicit immune activation in the subject or patient.
1006411 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of T
cell response in the subject or patient. In some instances, the level of T
cell response elicited by
the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower
compared to the level of T cell response produced by the administration of
immunogenic cells In
some embodiments, the administered population of hypoimmunogenic cells fails
to elicit a T cell
response to the cells in the subject or patient.
186
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00642] In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of NK
cell response in the subject or patient. In some instances, the level of NK
cell response elicited
by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower
compared to the level of NK cell response produced by the administration of
immunogenic cells.
In some embodiments, the administered population of hypoimmunogenic cells
fails to elicit an
NK cell response to the cells in the subject or patient.
[00643] In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
macrophage engulfment in the subject or patient. In some instances, the level
of NK cell
response elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or
99% lower compared to the level of macrophage engulfment produced by the
administration of
immunogenic cells. In some embodiments, the administered population of
hypoimmunogenic
cells fails to elicit macrophage engulfment of the cells in the subject or
patient.
[00644] In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
systemic TH1 activation in the subject or patient In some instances, the level
of systemic TH1
activation elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or
99% lower compared to the level of systemic TH1 activation produced by the
administration of
immunogenic cells. In some embodiments, the administered population of
hypoimmunogenic
cells fails to elicit systemic TH1 activation in the subject or patient.
1006451 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of NK
cell killing in the subject or patient. In some instances, the level of NK
cell killing elicited by the
cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower
compared to
the level of NK cell killing produced by the administration of immunogenic
cells. In some
187
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, the administered population of hypoimmunogenic cells fails to
elicit NK cell
killing in the subject or patient.
1006461 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
immune activation of peripheral blood mononuclear cells (PBMCs) in the subject
or patient. In
some instances, the level of immune activation of PBMCs elicited by the cells
is at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower compared to the
level of
immune activation of PBMCs produced by the administration of immunogenic
cells. In some
embodiments, the administered population of hypoimmunogenic cells fails to
elicit immune
activation of PBMCs in the subject or patient.
1006471 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
donor-specific IgG antibodies in the subject or patient. In some instances,
the level of donor-
specific IgG antibodies elicited by the cells is at least 5%, 10%, 15%, 20%,
25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, or 99% lower compared to the level of donor-specific IgG
antibodies produced
by the administration of immunogenic cells. In some embodiments, the
administered population
of hypoimmunogenic cells fails to elicit donor-specific IgG antibodies in the
subject or patient.
1006481 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
donor-specific IgM antibodies in the subject or patient. In some instances,
the level of donor-
specific IgM antibodies elicited by the cells is at least 5%, 10%, 15%, 20%,
25%, 30%, 35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, or 99% lower compared to the level of donor-specific IgM
antibodies produced
by the administration of immunogenic cells. In some embodiments, the
administered population
of hypoimmunogenic cells fails to elicit donor-specific IgM antibodies in the
subject or patient.
1006491 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of IgM
and IgG antibody production in the subject or patient. In some instances, the
level of IgM and
IgG antibody production elicited by the cells is at least 5%, 10%, 15%, 20%,
25%, 30%, 35%,
188
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%,
96%, 97%, 98%, or 99% lower compared to the level of IgM and IgG antibody
production
produced by the administration of immunogenic cells. In some embodiments, the
administered
population of hypoimmunogenic cells fails to elicit IgM and IgG antibody
production in the
subject or patient.
1006501 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
cytotoxic T cell killing in the subject or patient. In some instances, the
level of cytotoxic T cell
killing elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or
99% lower compared to the level of cytotoxic T cell killing produced by the
administration of
immunogenic cells. In some embodiments, the administered population of
hypoimmunogenic
cells fails to elicit cytotoxic T cell killing in the subject or patient.
1006511 In some embodiments, the administered population of hypoimmunogenic
cells such as
hypoimmunogenic differentiated cells and CAR-T cells elicits a decreased or
lower level of
complement-dependent cytotoxicity (CDC) in the subject or patient. In some
instances, the level
of CDC elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or
99% lower compared to the level of CDC produced by the administration of
immunogenic cells.
In some embodiments, the administered population of hypoimmunogenic cells
fails to elicit CDC
in the subject or patient.
S. Therapeutic Cells from Primary T Cells
1006521 Provided herein are hypoimmunogenic cells including, but not limited
to, primary T
cells that evade immune recognition. In some embodiments, the engineered
and/or
hypoimmunogenic cells are produced (e.g., generated, cultured, or derived)
from T cells such as
primary T cells. In some instances, primary T cells are obtained (e.g.,
harvested, extracted,
removed, or taken) from a subject or an individual. In some embodiments,
primary T cells are
produced from a pool of T cells such that the T cells are from one or more
subjects (e.g., one or
more human including one or more healthy humans). In some embodiments, the
pool of primary
T cells is from 1-100, 1-50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or
more, 5 or more, 10
189
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more
subjects. In some
embodiments, the donor subject is different from the patient (e.g., the
recipient that is
administered the therapeutic cells). In some embodiments, the pool of T cells
do not include
cells from the patient. In some embodiments, one or more of the donor subjects
from which the
pool of T cells is obtained are different from the patient.
[00653] In some embodiments, the engineered and/or hypoimmunogenic cells do
not activate an
innate and/or an adaptive immune response in the patient (e.g., recipient upon
administration).
Provided are methods of treating a disorder by administering a population of
hypoimmunogenic
cells to a subject (e.g., recipient) or patient in need thereof. In some
embodiments, the
engineered and/or hypoimmunogenic cells described herein comprise T cells
engineered (e.g.,
are modified) to express a chimeric antigen receptor including but not limited
to a chimeric
antigen receptor described herein. In some instances, the T cells are
populations or
subpopulations of primary T cells from one or more individuals. In some
embodiments, the T
cells described herein such as the engineered or modified T cells comprise
reduced expression of
an endogenous T cell receptor.
[00654] In some embodiments, the present disclosure is directed to
hypoimmunogenic primary
T cells that overexpress CD47 and CARs, and have reduced expression of one or
more Y
chromosome genes and reduced expression or lack expression of one or more MI-
IC class I
and/or MHC class II human leukocyte antigen molecules and have reduced
expression or lack
expression of TCR complex molecules. The cells outlined herein overexpress
CD47 and CARs
and evade immune recognition. In some embodiments, the primary T cells display
reduced
expression of one or more Y chromosome genes and reduced levels or activity of
MHC class I
antigen molecules, MEC class II antigen molecules, and/or TCR complex
molecules. In certain
embodiments, the primary T cells overexpress CD47 and CARs and harbor a
genetic
modification in the PCDH11Y gene. In certain embodiments, the primary T cells
overexpress
CD47 and CARs and harbor a genetic modification in the NLGN4Y gene. In certain

embodiments, the primary T cells overexpress CD47 and CARs and harbor a
genetic
modification in the PCDH11Y gene and a genetic modification in the NLGN4Y
gene. In certain
embodiments, primary T cells overexpress CD47 and CARs and harbor a genomic
modification
in the B2M gene. In some embodiments, T cells overexpress CD47 and CARs and
harbor a
genomic modification in the CIITA gene. In some embodiments, primary T cells
overexpress
190
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CD47 and CARs and harbor a genomic modification in the TRAC gene. In some
embodiments,
primary T cells overexpress CD47 and CARs and harbor a genomic modification in
the TRB
gene. In some embodiments, T cells overexpress CD47 and CARs and harbor
genomic
modifications in one or more of the following genes: the PCDH11Y, NLGN4Y, B2M,
CIITA,
TRAC and TRB genes
1006551 Exemplary T cells of the present disclosure are selected from the
group consisting of
cytotoxic T cells, helper T cells, memoiy T cells, central memory T cells,
effector memory T
cells, effector memory RA T cells, regulatory T cells, tissue infiltrating
lymphocytes, and
combinations thereof. In certain embodiments, the T cells express CCR7, CD27,
CD28, and
CD45RA. In some embodiments, the central T cells express CCR7, CD27, CD28, and
CD45RO.
In other embodiments, the effector memory T cells express PD-1, CD27, CD28,
and CD45RO.
In other embodiments, the effector memory RA T cells express PD-1, CD57, and
CD45RA.
1006561 In some embodiments, the T cell is a modified (e.g., an engineered) T
cell. In some
cases, the modified T cell comprise a modification causing the cell to express
at least one
chimeric antigen receptor that specifically binds to an antigen or epitope of
interest expressed on
the surface of at least one of a damaged cell, a dysplastic cell, an infected
cell, an immunogenic
cell, an inflamed cell, a malignant cell, a metaplastic cell, a mutant cell,
and combinations
thereof. In other cases, the modified T cell comprise a modification causing
the cell to express at
least one protein that modulates a biological effect of interest in an
adjacent cell, tissue, or organ
when the cell is in proximity to the adjacent cell, tissue, or organ. Useful
modifications to
primary T cells are described in detail in US2016/0348073 and W02020/018620,
the disclosures
of which are incorporated herein in their entireties.
1006571 In some embodiments, the engineered and/or hypoimmunogenic cells
described herein
comprise T cells that are engineered (e.g., are modified) to express a
chimeric antigen receptor
including but not limited to a chimeric antigen receptor described herein. In
some instances, the
T cells are populations or subpopulations of primary T cells from one or more
individuals. In
some embodiments, the T cells described herein such as the engineered or
modified T cells
include reduced expression of an endogenous T cell receptor. In some
embodiments, the T cells
described herein such as the engineered or modified T cells include reduced
expression of
cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). In other embodiments,
the T cells
described herein such as the engineered or modified T cells include reduced
expression of
191
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
programmed cell death (PD-1). In certain embodiments, the T cells described
herein such as the
engineered or modified T cells include reduced expression of CTLA-4 and PD-1.
Methods of
reducing or eliminating expression of CTLA-4, PD-1 and both CTLA-4 and PD-1
can include
any recognized by those skilled in the art, such as but not limited to,
genetic modification
technologies that utilize rare-cutting endonucleases and RNA silencing or RNA
interference
technologies. Non-limiting examples of a rare-cutting endonuclease include any
Cas protein,
TALEN, zinc finger nuclease, meganuclease, and homing endonuclease. In some
embodiments,
an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a
chimeric antigen
receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at
a CTLA-4 and/or
PD-1 gene locus.
[00658] In some embodiments, the T cells described herein such as the
engineered or modified
T cells include enhanced expression of PD-Li.
[00659] In some embodiments, the hypoimmunogenic T cell includes a
polynucleotide encoding
a CAR, wherein the polynucleotide is inserted in a genomic locus. In some
embodiments, the
polynucleotide is inserted into a safe harbor or target locus, such as but not
limited to, an
AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1

(also known as CD91), HIVIGB1, ABO, RHD, FUT1, or KDM5D gene locus. In some
embodiments, the polynucleotide is inserted in a B2M, CIITA, TRAC, TRB, PD-1
or CTLA-4
gene.
[00660] In some embodiments, the hypoimmunogenic T cell includes a
polynucleotide encoding
a CAR that is expressed in a cell using an expression vector. In some
embodiments, the CAR is
introduced to the cell using a viral expression vector that mediates
integration of the CAR
sequence using an into the genome of the cell. For example, the expression
vector for expressing
the CAR in a cell comprises a polynucleotide sequence encoding the CAR. The
expression
vector can be an inducible expression vector. The expression vector can be a
viral vector, such
as but not limited to, a lentiviral vector.
[00661] Hypoimmunogenic T cells provided herein are useful for the treatment
of suitable
cancers including, but not limited to, B cell acute lymphoblastic leukemia (B-
ALL), diffuse large
B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian
cancer, colorectal
cancer, lung cancer, non-small cell lung cancer, acute myeloid lymphoid
leukemia, multiple
192
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma,
glioblastoma,
neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and
bladder cancer.
T. Therapeutic Cells Differentiated from Hypoimmunogenic Pluripotent Stem
Cells
1006621 Provided herein are hypoimmunogenic cells including, cells derived
from pluripotent
stem cells, that evade immune recognition. In some embodiments, the cells do
not activate an
innate and/or an adaptive immune response in the patient or subject (e.g.,
recipient upon
administration). Provided are methods of treating a disorder comprising repeat
dosing of a
population of hypoimmunogenic cells to a recipient subject in need thereof.
1006631 In some embodiments, the pluripotent stem cell and any cell
differentiated from such a
pluripotent stem cell is modified to exhibit reduced expression of one or more
Y chromosome
genes and reduced expression of MHC class I human leukocyte antigen molecules.
In other
embodiments, the pluripotent stem cell and any cell differentiated from such a
pluripotent stem
cell is modified to exhibit reduced expression of one or more Y chromosome
genes and reduced
expression of MHC class II human leukocyte antigen molecules. In certain
embodiments, the
pluripotent stem cell and any cell differentiated from such a pluripotent stem
cell is modified to
exhibit reduced expression of one or more Y chromosome genes and reduced
expression of TCR
complexes. In some embodiments, the pluripotent stem cell and any cell
differentiated from such
a pluripotent stem cell is modified to exhibit reduced expression of one or
more Y chromosome
genes and reduced expression of MHC class I and II human leukocyte antigen
molecules. In
some embodiments, the pluripotent stem cell and any cell differentiated from
such a pluripotent
stem cell is modified to exhibit reduced expression of one or more Y
chromosome genes and
reduced expression of MHC class I and II human leukocyte antigen molecules and
TCR
complexes.
1006641 In some embodiments, the pluripotent stem cell and any cell
differentiated from such a
pluripotent stem cell is modified to exhibit reduced expression of one or more
Y chromosome
genes and reduced expression of MHC class I and/or II human leukocyte antigen
molecules and
exhibit increased CD47 expression. In some instances, the cell overexpresses
CD47 by
harboring one or more CD47 transgenes. In some embodiments, the pluripotent
stem cell and
any cell differentiated from such a pluripotent stem cell is modified to
exhibit reduced expression
of one or more Y chromosome genes and reduced expression of MHC class I and II
human
193
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
leukocyte antigen molecules and exhibit increased CD47 expression. In some
embodiments, the
pluripotent stem cell and any cell differentiated from such a pluripotent stem
cell is modified to
exhibit reduced expression of one or more Y chromosome genes and reduced
expression of
MHC class I and II human leukocyte antigen molecules and TCR complexes and
exhibit
increased CD47 expression.
1006651 In some embodiments, the pluripotent stem cell and any cell
differentiated from such a
pluripotent stem cell is modified to exhibit reduced expression of one or more
Y chromosome
genes and reduced expression of MHC class I and/or II human leukocyte antigen
molecules, to
exhibit increased CD47 expression, and to exogenously express a chimeric
antigen receptor. In
some instances, the cell overexpresses CD47 polypeptides by harboring one or
more CD47
transgenes. In some instances, the cell overexpresses CAR polypeptides by
harboring one or
more CAR transgenes. In some embodiments, the pluripotent stem cell and any
cell
differentiated from such a pluripotent stem cell is modified to exhibit
reduced expression of one
or more Y chromosome genes and reduced expression of MHC class I and II human
leukocyte
antigen molecules, exhibit increased CD47 expression, and to exogenously
express a chimeric
antigen receptor. In some embodiments, the pluripotent stem cell and any cell
differentiated
from such a pluripotent stem cell is modified to exhibit reduced expression of
one or more Y
chromosome genes and reduced expression of MHC class I and II human leukocyte
antigen
molecules and TCR complexes, to exhibit increased CD47 expression, and to
exogenously
express a chimeric antigen receptor.
1006661 Such pluripotent stem cells are hypoimmunogenic stem cells. Such
differentiated cells
are hypoimmunogenic cells.
1006671 Any of the pluripotent stem cells described herein can be
differentiated into any cells of
an organism and tissue. In some embodiments, the cells exhibit reduced
expression of one or
more Y chromosome genes, reduced expression of MEW class I and/or II human
leukocyte
antigen molecules, and reduced expression of TCR complexes. In some instances,
expression of
the one or more Y chromosome genes is reduced compared to unmodified or wild-
type cell of
the same cell type. In some instances, expression of MHC class I and/or II
human leukocyte
antigen molecules is reduced compared to unmodified or wild-type cell of the
same cell type. In
some instances, expression of TCR complexes is reduced compared to unmodified
or wild-type
cell of the same cell type. In some embodiments, the cells exhibit increased
CD47 expression. In
194
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some instances, expression of CD47 is increased in cells encompassed by the
present disclosure
as compared to unmodified or wild-type cells of the same cell type. In some
embodiments, the
cells exhibit exogenous CAR expression. Methods for reducing levels of MTIC
class I and/or II
human leukocyte antigen molecules and TCR complexes and increasing the
expression of CD47
and CARs are described herein.
1006681 In some embodiments, the cells used in the methods described herein
evade immune
recognition and responses when administered to a patient (e.g., recipient
subject). The cells can
evade killing by immune cells in vitro and in vivo. In some embodiments, the
cells evade killing
by macrophages and NK cells. In some embodiments, the cells are ignored by
immune cells or a
subject's immune system. In other words, the cells administered in accordance
with the methods
described herein are not detectable by immune cells of the immune system. In
some
embodiments, the cells are cloaked and therefore avoid immune rejection.
1006691 Methods of determining whether a pluripotent stem cell and any cell
differentiated from
such a pluripotent stem cell evades immune recognition include, but are not
limited to, IFN-y
Elispot assays, microglia killing assays, cell engraftment animal models,
cytokine release assays,
ELISAs, killing assays using bioluminescence imaging or chromium release assay
or a real-time,
quantitative microelectronic biosensor system for cell analysis (xCELLigence
RTCA system,
Agilent), mixed-lymphocyte reactions, immunofluorescence analysis, etc.
1006701 Therapeutic cells outlined herein are useful to treat a disorder such
as, but not limited
to, a cancer, a genetic disorder, a chronic infectious disease, an autoimmune
disorder, a
neurological disorder, and the like.
1. T Lymphocytes Differentiated from Hypoimmunogenic Pluripotent Cells
1006711 Provided herein, T lymphocytes (T cells, including primary T cells)
are derived from
the HIP cells described herein (e.g., hypoimmunogenic iPSCs). Methods for
generating T cells,
including CAR-T cells, from pluripotent stem cells (e.g., iPSCs) are
described, for example, in
Iriguchi et al., Nature Communications 12, 430 (2021); Themeli et al., Cell
Stem Cell,
16(4):357-366 (2015); Themeli et al., Nature Biotechnology 31:928-933 (2013).
1006721 T lymphocyte derived hypoimmunogenic cells include, but are not
limited to, primary
T cells that evade immune recognition. In some embodiments, the
hypoimmunogenic cells are
produced (e.g., generated, cultured, or derived) from T cells such as primary
T cells. In some
195
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
instances, primary T cells are obtained (e.g., harvested, extracted, removed,
or taken) from a
subject or an individual. In some embodiments, primary T cells are produced
from a pool of T
cells such that the T cells are from one or more subjects (e.g., one or more
human including one
or more healthy humans). In some embodiments, the pool of primary T cells is
from 1-100, 1-
50, 1-20, 1-10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or
more, 20 or more, 30
or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments,
the donor
subject is different from the patient (e.g., the recipient that is
administered the therapeutic cells).
In some embodiments, the pool of T cells does not include cells from the
patient. In some
embodiments, one or more of the donor subjects from which the pool of T cells
is obtained are
different from the patient.
1006731 In some embodiments, the hypoimmunogenic cells do not activate an
immune response
in the patient (e.g., recipient upon administration). Provided are methods of
treating a disorder
by administering a population of hypoimmunogenic cells to a subject (e.g.,
recipient) or patient
in need thereof. In some embodiments, the hypoimmunogenic cells described
herein comprise T
cells engineered (e.g., are modified) to express a chimeric antigen receptor
including but not
limited to a chimeric antigen receptor described herein. In some instances,
the T cells are
populations or subpopulations of primary T cells from one or more individuals.
In some
embodiments, the T cells described herein such as the engineered or modified T
cells comprise
reduced expression of an endogenous T cell receptor.
1006741 In some embodiments, the HIP-derived T cell includes a chimeric
antigen receptor
(CAR). Any suitable CAR can be included in the hyHIP-derived T cell, including
the CARs
described herein. In some embodiments, the hypoimmunogenic induced pluripotent
stem cell-
derived T cell includes a polynucleotide encoding a CAR, wherein the
polynucleotide is inserted
in a genomic locus. In some embodiments, the polynucleotide is inserted into a
safe harbor or
target locus. In some embodiments, the polynucleotide is inserted in a B2M,
CIITA, TRAC,
TRB, PD-1 or CTLA-4 gene. Any suitable method can be used to insert the CAR
into the
genomic locus of the hypoimmunogenic cell including the gene editing methods
described herein
(e.g., a CRISPR/Cas system).
1006751 HIP-derived T cells provided herein are useful for the treatment of
suitable
autoimmune diseases/disorders and/or inflammatory diseases/disorders
including, but not limited
196
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
to, diseases of the nervous, gastrointestinal, and endocrine systems, as well
as skin and other
connective tissues, eyes, blood and blood vessels. Examples of autoimmune
diseases include, but
are not limited to Hashimoto's thyroiditis, Systemic lupus erythematosus,
Sjogren's syndrome,
Graves' disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis, MS
associated with EBV
infection, Myasthenia gravis and Diabetes.
2. NK Cells Derived from Hypoimmunogenic Pluripotent Cells
[00676] Provided herein, natural killer (NK) cells are derived from the HIP
cells described
herein (e.g., hypoimmunogenic iPSCs).
[00677] NK cells (also defined as 'large granular lymphocytes') represent a
cell lineage
differentiated from the common lymphoid progenitor (which also gives rise to B
lymphocytes
and T lymphocytes). Unlike T-cells, NK cells do not naturally comprise CD3 at
the plasma
membrane. Importantly, NK cells do not express a TCR and typically also lack
other antigen-
specific cell surface receptors (as well as TCRs and CD3, they also do not
express
immunoglobulin B-cell receptors, and instead typically express CD16 and CD56).
NK cell
cytotoxic activity does not require sensitization but is enhanced by
activation with a variety of
cytokines including IL-2. NK cells are generally thought to lack appropriate
or complete
signaling pathways necessary for antigen-receptor-mediated signaling, and thus
are not thought
to be capable of antigen receptor-dependent signaling, activation and
expansion. NK cells are
cytotoxic, and balance activating and inhibitory receptor signaling to
modulate their cytotoxic
activity. For instance, NK cells expressing CD16 may bind to the Fc domain of
antibodies bound
to an infected cell, resulting in NK cell activation. By contrast, activity is
reduced against cells
expressing high levels of MEW class I proteins/molecules. On contact with a
target cell NK cells
release proteins such as perforin, and enzymes such as proteases (granzymes).
Perforin can form
pores in the cell membrane of a target cell, inducing apoptosis or cell lysis.
[00678] There are a number of techniques that can be used to generate NK
cells, including
CAR-NK-cells, from pluripotent stem cells (e.g., iPSC); see, for example, Zhu
et al., Methods
Mol Biol. 2019; 2048:107-119; Knorr et al, Stem Cells Tratisl Med. 2013
2(4):274-83. doi:
10.5966/sctm .2012-0084; Zeng et al. õS'tem Cell Reports. 2017 Dec
12;9(6):1796-1812; Ni et al.,
Methods' Mol Biol. 2013;1029:33-41; Bernareggi et al., Exp Hematol. 2019 71:13-
23; Shankar et
al., Stem Cell Res Ther. 2020;11(1):234, all of which are incorporated herein
by reference in
197
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
their entirety and specifically for the methodologies and reagents for
differentiation.
Differentiation can be assayed as is known in the art, generally by evaluating
the presence of NK
cell associated and/or specific markers, including, but not limited to, CD56,
KIRs, CD16,
NKp44, NKp46, NKG2D, TRAIL, CD122, CD27, CD244, NK1 1, NKG2A/C, NCR1, Ly49,
CD49b, CD11b, KLRG1, CD43, CD62L, and/or CD226
3. Cardiac Cells
1006791 Provided herein are cardiac cell types differentiated from
hypoimmunogenic induced
pluripotent (HIP) cells for subsequent transplantation or engraftment into
subjects (e.g.,
recipients). As will be appreciated by those in the art, the methods for
differentiation depend on
the desired cell type using known techniques. Exemplary cardiac cell types
include, but are not
limited to, a cardiomyocyte, nodal cardiomyocyte, conducting cardiomyocyte,
working
cardiomyocyte, cardiomyocyte precursor cell, cardiomyocyte progenitor cell,
cardiac stem cell,
cardiac muscle cell, atrial cardiac stem cell, ventricular cardiac stem cell,
epicardial cell,
hematopoietic cell, vascular endothelial cell, endocardial endothelial cell,
cardiac valve
interstitial cell, cardiac pacemaker cell, and the like.
1006801 In some embodiments, cardiac cells described herein are administered
to a recipient
subject to treat a cardiac disorder selected from the group consisting of
pediatric
cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy,
hypertrophic
cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy,
peripartum
cardiomyopathy, inflammatory cardiomyopathy, idiopathic cardiomyopathy, other
cardiomyopathy, myocardial ischemic reperfusion injury, ventricular
dysfunction, heart failure,
congestive heart failure, coronary artery disease, end-stage heart disease,
atherosclerosis,
ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial
inflammation,
cardiovascular disease, myocardial infarction, myocardial ischemia, congestive
heart failure,
myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia,
vascular disease,
acquired heart disease, congenital heart disease, atherosclerosis, coronary
artery disease,
dysfunctional conduction systems, dysfunctional coronary arteries, pulmonary
hypertension,
cardiac arrhythmias, muscular dystrophy, muscle mass abnormality, muscle
degeneration,
myocarditis, infective myocarditis, drug- or toxin-induced muscle
abnormalities, hypersensitivity
myocarditis, and autoimmune endocarditis.
198
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00681] Accordingly, provided herein are methods for the treatment and
prevention of a cardiac
injury or a cardiac disease or disorder in a subject in need thereof. The
methods described herein
can be used to treat, ameliorate, prevent or slow the progression of a number
of cardiac diseases
or their symptoms, such as those resulting in pathological damage to the
structure and/or
function of the heart. The terms "cardiac disease," "cardiac disorder," and
"cardiac injury," are
used interchangeably herein and refer to a condition and/or disorder relating
to the heart,
including the valves, endothelium, infarcted zones, or other components or
structures of the
heart. Such cardiac diseases or cardiac-related disease include, but are not
limited to, myocardial
infarction, heart failure, cardiomyopathy, congenital heart defect, heart
valve disease or
dysfunction, endocarditis, rheumatic fever, mitral valve prolapse, infective
endocarditis,
hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis,
cardiomegaly, and/or mitral
insufficiency, among others.
[00682] In some embodiments, the cardiomyocyte precursor includes a cell that
is capable
giving rise to progeny that include mature (end-stage) cardiomyocytes.
Cardiomyocyte precursor
cells can often be identified using one or more markers selected from GATA-4,
Nkx2.5, and the
1VIEF-2 family of transcription factors. In some instances, cardiomyocytes
refer to immature
cardiomyocytes or mature cardiomyocytes that express one or more markers
(sometimes at least
2, 3, 4 or 5 markers) from the following list: cardiac troponin I (cTn1),
cardiac troponin T
(cTnT), sarcomeric myosin heavy chain (MT-IC), GATA-4, Nkx2.5, N-cadherin, 132-

adrenoceptor, ANF, the MEF-2 family of transcription factors, creatine kinase
MB (CK-MB),
myoglobin, and atrial natriuretic factor (ANF). In some embodiments, the
cardiac cells
demonstrate spontaneous periodic contractile activity. In some cases, when
that cardiac cells are
cultured in a suitable tissue culture environment with an appropriate Ca2+
concentration and
electrolyte balance, the cells can be observed to contract in a periodic
fashion across one axis of
the cell, and then release from contraction, without having to add any
additional components to
the culture medium. In some embodiments, the cardiac cells are hypoimmunogenic
cardiac cells.
[00683] In some embodiments, the method of producing a population of
hypoimmunogenic
cardiac cells from a population of hypoimmunogenic induced pluripotent stem
cells by in vitro
differentiation comprises: (a) culturing a population of hypoimmunogenic
induced pluripotent
stem cells in a culture medium comprising a GSK inhibitor; (b) culturing the
population of
hypoimmunogenic induced pluripotent stem cells in a culture medium comprising
a WNT
199
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
antagonist to produce a population of pre-cardiac cells; and (c) culturing the
population of pre-
cardiac cells in a culture medium comprising insulin to produce a population
of hypoimmune
cardiac cells. In some embodiments, the GSK inhibitor is CHIR-99021, a
derivative thereof, or a
variant thereof. In some instances, the GSK inhibitor is at a concentration
ranging from about 2
mM to about 10 mM. In some embodiments, the WNT antagonist is IWR1, a
derivative thereof,
or a variant thereof. In some instances, the WNT antagonist is at a
concentration ranging from
about. 2 inM to about. 10 inM.
1006841 In some embodiments, the population of hypoimmunogenic cardiac cells
is isolated
from non-cardiac cells. In some embodiments, the isolated population of
hypoimmunogenic
cardiac cells are expanded prior to administration. In certain embodiments,
the isolated
population of hypoimmunogenic cardiac cells are expanded and cryopreserved
prior to
administration.
1006851 Other useful methods for differentiating induced pluripotent stem
cells or pluripotent
stem cells into cardiac cells are described, for example, in US2017/0152485;
US2017/0058263;
US2017/0002325; US2016/0362661; US2016/0068814; US9,062,289; US7,897,389; and
US7,452,718. Additional methods for producing cardiac cells from induced
pluripotent stem
cells or pluripotent stem cells are described in, for example, Xu et al, Stem
Cells and
Development, 2006, 15(5): 631-9, Burridge et al, Cell Stem Cell, 2012, 10: 16-
28, and Chen et
al, Stem Cell Res, 2015, 15(2):365-375.
1006861 In various embodiments, hypoimmunogenic cardiac cells can be cultured
in culture
medium comprising a BMP pathway inhibitor, a WNT signaling activator, a WNT
signaling
inhibitor, a WNT agonist, a WNT antagonist, a Src inhibitor, a EGFR inhibitor,
a PCK activator,
a cytokine, a growth factor, a cardiotropic agent, a compound, and the like.
1006871 The WNT signaling activator includes, but is not limited to,
CHIR99021. The PCK
activator includes, but is not limited to, PMA. The WNT signaling inhibitor
includes, but is not
limited to, a compound selected from KY02111, S03031 (KY01-I), S02031 (KY024),
and
S03042 (KY034), and XAV939. The Src inhibitor includes, but is not limited to,
A419259. The
EGFR inhibitor includes, but is not limited to, AG1478.
1006881 Non-limiting examples of an agent for generating a cardiac cell from
an iPSC include
activin A, BMP4, Wnt3a, VEGF, soluble frizzled protein, cyclosporin A,
angiotensin II,
phenylephrine, ascorbic acid, dimethylsulfoxide, 5-aza-2'-deoxycytidine, and
the like.
200
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1006891 The cells provided herein can be cultured on a surface, such as a
synthetic surface to
support and/or promote differentiation of hypoimmunogenic pluripotent cells
into cardiac cells.
In some embodiments, the surface comprises a polymer material including, but
not limited to, a
homopolymer or copolymer of selected one or more acrylate monomers. Non-
limiting examples
of acrylate monomers and methacrylate monomers include tetra(ethylene glycol)
diacrylate,
glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol)
diacrylate,
di(ethylene glycol) dimethacrylate, tetra(eillyiene glycol) dimethacrylate,
1,6-hexanediol
propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane
benzoate diacrylate,
trimethylolpropane eihoxylate (1 EO/QH) methyl, tricyclo[5.2.1.02,6] decane
dimethanol
diacrylate, neopentyl glycol ethoxylate diacrylate, and trimethylolpropane
triacrylate. Acrylate
synthesized as known in the art or obtained from a commercial vendor, such as
Polysciences,
Inc., Sigma Aldrich, Inc. and Sartomer, Inc.
1006901 The polymeric material can be dispersed on the surface of a support
material. Useful
support materials suitable for culturing cells include a ceramic substance, a
glass, a plastic, a
polymer or co-polymer, any combinations thereof, or a coating of one material
on another. In
some instances, a glass includes soda-lime glass, pyrex glass, vycor glass,
quartz glass, silicon,
or derivatives of these or the like.
1006911 In some instances, plastics or polymers including dendritic polymers
include poly(vinyl
chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-
maleic anhydride),
poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon
polymers,
polystyrenes, polypropylene, polyethyleneimine or derivatives of these or the
like. In some
instances, copolymers include poly(vinyl acetate-co-maleic anhydride),
poly(styrene-co-maleic
anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the
like.
1006921 The efficacy of cardiac cells prepared as described herein can be
assessed in animal
models for cardiac cryoinjury, which causes 55% of the left ventricular wall
tissue to become
scar tissue without treatment (Li et al, Ann. Thorac. Surg. 62:654, 1996;
Sakai et al, Ann.
Thorac. Surg. 8:2074, 1999, Sakai et al., Thorac. Cardiovasc. Surg. 118:715,
1999). Successful
treatment can reduce the area of the scar, limit scar expansion, and improve
heart function as
determined by systolic, diastolic, and developed pressure. Cardiac injury can
also be modeled
using an embolization coil in the distal portion of the left anterior
descending artery (Watanabe
201
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
et al., Cell Transplant. 7:239, 1998), and efficacy of treatment can be
evaluated by histology and
cardiac function.
[00693] In some embodiments, the administration comprises implantation into
the subject's
heart tissue, intravenous injection, intraarteri al injection, intracoronary
injection, intramuscular
injection, intraperitoneal injection, intramyocardial injection, trans-
endocardial injection, trans-
epicardial injection, or infusion.
[00694] In some embodiments, the patient administered the engineered cardiac
cells is also
administered a cardiac drug. Illustrative examples of cardiac drugs that are
suitable for use in
combination therapy include, but are not limited to, growth factors,
polynucleotides encoding
growth factors, angiogenic agents, calcium channel blockers, antihypertensive
agents, antimitotic
agents, inotropic agents, anti-atherogenic agents, anti-coagulants, beta-
blockers, anti-arhythmic
agents, anti-inflammatory agents, vasodilators, thrombolytic agents, cardiac
glycosides,
antibiotics, antiviral agents, antifungal agents, agents that inhibit
protozoans, nitrates,
angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor
antagonist, brain
natriuretic peptide (BNP); antineoplastic agents, steroids, and the like.
[00695] The effects of therapy according to the methods provided herein can be
monitored in a
variety of ways. For instance, an electrocardiogram (ECG) or holier monitor
can be utilized to
determine the efficacy of treatment. An ECG is a measure of the heart rhythms
and electrical
impulses, and is a very effective and non-invasive way to determine if therapy
has improved or
maintained, prevented, or slowed degradation of the electrical conduction in a
subject's heart.
The use of a holier monitor, a portable ECG that can be worn for long periods
of time to monitor
heart abnormalities, arrhythmia disorders, and the like, is also a reliable
method to assess the
effectiveness of therapy. An ECG or nuclear study can be used to determine
improvement in
ventricular function.
4. Neural Cells
[00696] Provided herein are different neural cell types differentiated from
hypoimmunogenic
induced pluripotent stem (HIP) cells that are useful for subsequent
transplantation or engraftment
into recipient subjects. As will be appreciated by those in the art, the
methods for differentiation
depend on the desired cell type using known techniques. Exemplary neural cell
types include,
202
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
but are not limited to, cerebral endothelial cells, neurons (e.g.,
dopaminergic neurons), glial cells,
and the like.
[00697] In some embodiments, differentiation of induced pluripotent stem cells
is performed by
exposing or contacting cells to specific factors which are known to produce a
specific cell
lineage(s), so as to target their differentiation to a specific, desired
lineage and/or cell type of
interest. In some embodiments, terminally differentiated cells display
specialized phenotypic
characteristics or features. In certain embodiments, the stem cells described
herein are
differentiated into a neuroectodermal, neuronal, neuroendocrine, dopaminergic,
cholinergic,
serotonergic (5-HT), glutamatergic, GABAergic, adrenergic, noradrenergic,
sympathetic
neuronal, parasympathetic neuronal, sympathetic peripheral neuronal, or glial
cell population. In
some instances, the glial cell population includes a microglial (e.g.,
amoeboid, ramified,
activated phagocytic, and activated non-phagocytic) cell population or a
macroglial (central
nervous system cell: astrocyte, oligodendrocyte, ependymal cell, and radial
glia; and peripheral
nervous system cell: Schwann cell and satellite cell) cell population, or the
precursors and
progenitors of any of the preceding cells.
[00698] Protocols for generating different types of neural cells arc described
in PCT
Application No. W02010144696, US Patent Nos. 9,057,053; 9,376,664; and
10,233,422.
Additional descriptions of methods for differentiating hypoimmunogenic
pluripotent cells can be
found, for example, in Deuse et al., Nature Biotechnology, 2019, 37, 252-258
and Han et al,,
Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446. Methods for determining
the effect of
neural cell transplantation in an animal model of a neurological disorder or
condition are
described in the following references: for spinal cord injury ¨ Curtis et al.,
Cell Stem Cell, 2018,
22, 941-950, for Parkinson's disease ¨ Kikuchi et al., Nature, 2017, 548.592-
596, for ALS ¨
Izrael et al., Stem Cell Research, 2018, 9(1):152 and Izrael et al.,
IntechOpen, DOT:
10.5772/intechopen.72862; for epilepsy ¨ Upadhya et al., PNAS, 2019,
116(1):287-296
5. Cerebral endothelial cells
[00699] In some embodiments, neural cells are administered to a subject to
treat Parkinson's
disease, Huntington disease, multiple sclerosis, other neurodegenerative
disease or condition,
attention deficit hyperactivity disorder (ADHD), Tourette Syndrome (TS),
schizophrenia,
psychosis, depression, other neuropsychiatric disorder. In some embodiments,
neural cells
203
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
described herein are administered to a subject to treat or ameliorate stroke.
In some
embodiments, the neurons and glial cells are administered to a subject with
amyotrophic lateral
sclerosis (ALS). In some embodiments, cerebral endothelial cells are
administered to alleviate
the symptoms or effects of cerebral hemorrhage. In some embodiments,
dopaminergic neurons
are administered to a patient with Parkinson's disease In some embodiments,
noradrenergic
neurons, GABAergic interneurons are administered to a patient who has
experienced an epileptic
seizure. In some embodiments, motor neurons, inter neurons, Schwalm cells,
oligodendi ocy les,
and microglia are administered to a patient who has experienced a spinal cord
injury.
1007001 In some embodiments, cerebral endothelial cells (ECs), precursors, and
progenitors
thereof are differentiated from pluripotent stem cells (e.g., induced
pluripotent stem cells) on a
surface by culturing the cells in a medium comprising one or more factors that
promote the
generation of cerebral ECs or neural cell. In some instances, the medium
includes one or more of
the following: CHIR-99021, VEGF, basic FGF (bFGF), and Y-27632. In some
embodiments, the
medium includes a supplement designed to promote survival and functionality
for neural cells.
1007011 In some embodiments, cerebral endothelial cells (ECs), precursors, and
progenitors
thereof are differentiated from pluripotent stem cells on a surface by
culturing the cells in an
unconditioned or conditioned medium. In some instances, the medium comprises
factors or
small molecules that promote or facilitate differentiation. In some
embodiments, the medium
comprises one or more factors or small molecules selected from the group
consisting of VEGR,
FGF, SDF-1, CHIR-99021, Y-27632, SB 431542, and any combination thereof. In
some
embodiments, the surface for differentiation comprises one or more
extracellular matrix proteins.
The surface can be coated with the one or more extracellular matrix proteins.
The cells can be
differentiated in suspension and then put into a gel matrix form, such as
matrigel, gelatin, or
fibrin/thrombin forms to facilitate cell survival. In some cases,
differentiation is assayed as is
known in the art, generally by evaluating the presence of cell-specific
markers.
1007021 In some embodiments, the cerebral endothelial cells express or secrete
a factor selected
from the group consisting of CD31, VE cadherin, and a combination thereof. In
certain
embodiments, the cerebral endothelial cells express or secrete one or more of
the factors selected
from the group consisting of CD31, CD34, CD45, CD117 (c-kit), CD146, CXCR4,
VEGF, SDF-
1, PDGF, GLUT-1, PECAM-1, eNOS, claudin-5, occludin, ZO-1, p-glycoprotein, von

Willebrand factor, VE-cadherin, low density lipoprotein receptor LDLR, low
density lipoprotein
204
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
receptor-related protein I LRP1, insulin receptor INSR, leptin receptor LEPR,
basal cell
adhesion molecule BCAM, transferrin receptor TFRC, advanced glycation
endproduct-specific
receptor AGER, receptor for retinol uptake STRA6, large neutral amino acids
transporter small
subunit 1 SLC7A5, excitatory amino acid transporter 3 SLC1A1, sodium-coupled
neutral amino
acid transporter 5 SLC38A5, solute carrier family 16 member 1 SLC16A1, ATP-
dependent
translocase ABCB1, ATP-ABCC2-binding cassette transporter ABCG2, multidrug
resistance-
associated protein 1 ABCC I, canaliculai multispecific organic anion
transporter 1 ABCC2,
multidrug resistance-associated protein 4 ABCC4, and multidrug resistance-
associated protein 5
ABCC5.
1007031 In some embodiments, the cerebral ECs are characterized with one or
more of the
features selected from the group consisting of high expression of tight
junctions, high electrical
resistance, low fenestration, small perivascular space, high prevalence of
insulin and transferrin
receptors, and high number of mitochondria.
1007041 In some embodiments, cerebral ECs are selected or purified using a
positive selection
strategy. In some instances, the cerebral ECs are sorted against an
endothelial cell marker such
as, but not limited to, CD31. In other words, CD31 positive cerebral ECs arc
isolated. In some
embodiments, cerebral ECs are selected or purified using a negative selection
strategy. In some
embodiments, undifferentiated or pluripotent stem cells are removed by
selecting for cells that
express a pluripotency marker including, but not limited to, TRA-1-60 and SSEA-
1.
6. Dopaminergic neurons
1007051 In some embodiments, hypoimmunogenic induced pluripotent stem
(HIP)cells
described herein are differentiated into dopaminergic neurons include neuronal
stem cells,
neuronal progenitor cells, immature dopaminergic neurons, and mature
dopaminergic neurons.
1007061 In some cases, the term "dopaminergic neurons" includes neuronal cells
which express
tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. In
some
embodiments, dopaminergic neurons secrete the neurotransmitter dopamine, and
have little or no
expression of dopamine hydroxylase. A dopaminergic (DA) neuron can express one
or more of
the following markers: neuron-specific enolase (NSE), 1-aromatic amino acid
decarboxylase,
vesicular monoamine transporter 2, dopamine transporter, Nurr-1, and dopamine-
2 receptor (D2
receptor). In certain cases, the term "neural stem cells" includes a
population of pluripotent cells
205
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
that have partially differentiated along a neural cell pathway and express one
or more neural
markers including, for example, nestin. Neural stem cells may differentiate
into neurons or glial
cells (e.g., astrocytes and oligodendrocytes). The term "neural progenitor
cells" includes cultured
cells which express FOXA2 and low levels of b-tubulin, but not tyrosine
hydroxyl ase. Such
neural progenitor cells have the capacity to differentiate into a variety of
neuronal subtypes;
particularly a variety of dopaminergic neuronal subtypes, upon culturing the
appropriate factors,
such as those described herein.
[00707] In some embodiments, the DA neurons derived from hypoimmunogenic
induced
pluripotent stem (HIP) cells are administered to a patient, e.g., human
patient to treat a
neurodegenerative disease or condition. In some cases, the neurodegenerative
disease or
condition is selected from the group consisting of Parkinson's disease,
Huntington disease, and
multiple sclerosis. In other embodiments, the DA neurons are used to treat or
ameliorate one or
more symptoms of a neuropsychiatric disorder, such as attention deficit
hyperactivity disorder
(ADHD), Tourette Syndrome (TS), schizophrenia, psychosis, and depression. In
yet other
embodiments, the DA neurons are used to treat a patient with impaired DA
neurons.
[00708] In some embodiments, DA neurons, precursors, and progenitors thereof
are
differentiated from pluripotent stem cells by culturing the stem cells in
medium comprising one
or more factors or additives. Useful factors and additives that promote
differentiation, growth,
expansion, maintenance, and/or maturation of DA neurons include, but are not
limited to, Wntl,
FGF2, FGF8, FGF8a, sonic hedgehog (SHIT), brain derived neurotrophic factor
(BDNF),
transforming growth factor a (TGF-a), TGF-b, interleukin 1 beta, glial cell
line-derived
neurotrophic factor (GDNF), a GSK-3 inhibitor (e.g., CHIR-99021), a TGF-b
inhibitor (e.g., SB-
431542), B-27 supplement, dorsomorphin, purmorphamine, noggin, retinoic acid,
cAMP,
ascorbic acid, neurturin, knockout serum replacement, N-acetyl cysteine, c-kit
ligand, modified
forms thereof, mimics thereof, analogs thereof, and variants thereof In some
embodiments, the
DA neurons are differentiated in the presence of one or more factors that
activate or inhibit the
WNT pathway, NOTCH pathway, SHH pathway, BMP pathway, FGF pathway, and the
like.
Differentiation protocols and detailed descriptions thereof are provided in,
e.g., US9,968,637,
US7,674,620, Kim et al, Nature, 2002, 418,50-56; Bjorklund et al, PNAS, 2002,
99(4), 2344-
2349; Grow et al., Stem Cells Transl Med. 2016, 5(9): 1133-44, and Cho et al,
PNAS, 2008,
206
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
105:33 92-33 97, the disclosures in their entirety including the detailed
description of the
examples, methods, figures, and results are herein incorporated by reference.
1007091 In some embodiments, the population of hypoimmunogenic dopaminergic
neurons is
isolated from non-neuronal cells. In some embodiments, the isolated population
of
hypoimmunogenic dopaminergic neurons are expanded prior to administration. In
certain
embodiments, the isolated population of hypoimmunogenic dopaminergic neurons
are expanded
and ciyopiesei ved prior to administration.
1007101 To characterize and monitor DA differentiation and assess the DA
phenotype,
expression of any number of molecular and genetic markers can be evaluated.
For example, the
presence of genetic markers can be determined by various methods known to
those skilled in the
art. Expression of molecular markers can be determined by quantifying methods
such as, but not
limited to, qPCR-based assays, immunoassays, immunocytochemistry assays,
immunoblotting
assays, and the like. Exemplary markers for DA neurons include, but are not
limited to, TH, b-
tubulin, paired box protein (Pax6), insulin gene enhancer protein (Is11),
nestin, diaminobenzidine
(DAB), G protein-activated inward rectifier potassium channel 2 (GIRK2),
microtubule-
associated protein 2 (MAP-2), NURR1, dopamine transporter (DAT), forkhcad box
protein A2
(FOXA2), FOX3, doublecortin, and LIM homeobox transcription factor 1-beta
(LMX1B), and
the like. In some embodiments, the DA neurons express one or more of the
markers selected
from corin, FOXA2, TuJ1, NURR1, and any combination thereof.
1007111 In some embodiments, DA neurons are assessed according to cell
electrophysiological
activity. The electrophysiology of the cells can be evaluated by using assays
knowns to those
skilled in the art. For instance, whole-cell and perforated patch clamp,
assays for detecting
electrophysiological activity of cells, assays for measuring the magnitude and
duration of action
potential of cells, and functional assays for detecting dopamine production of
DA cells.
1007121 In some embodiments, DA neuron differentiation is characterized by
spontaneous
rhythmic action potentials, and high-frequency action potentials with spike
frequency adaption
upon injection of depolarizing current. In other embodiments, DA
differentiation is characterized
by the production of dopamine. The level of dopamine produced is calculated by
measuring the
width of an action potential at the point at which it has reached half of its
maximum amplitude
(spike half-maximal width).
207
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007131 In some embodiments, the differentiated DA neurons are transplanted
either
intravenously or by injection at particular locations in the patient. In some
embodiments, the
differentiated DA cells are transplanted into the substantia nigra
(particularly in or adjacent of
the compact region), the ventral tegmental area (VTA), the caudate, the
putamen, the nucleus
accumbens, the subthalamic nucleus, or any combination thereof, of the brain
to replace the DA
neurons whose degeneration resulted in Parkinson's disease. The differentiated
DA cells can be
injected into the target area as a cell suspension. Alternatively, the
differentiated DA cells can be
embedded in a support matrix or scaffold when contained in such a delivery
device. In some
embodiments, the scaffold is biodegradable. In other embodiments, the scaffold
is not
biodegradable. The scaffold can comprise natural or synthetic (artificial)
materials.
1007141 The delivery of the DA neurons can be achieved by using a suitable
vehicle such as, but
not limited to, liposomes, microparticles, or microcapsules. In other
embodiments, the
differentiated DA neurons are administered in a pharmaceutical composition
comprising an
isotonic excipient. The pharmaceutical composition is prepared under
conditions that are
sufficiently sterile for human administration. In some embodiments, the DA
neurons
differentiated from HIP cells arc supplied in the form of a pharmaceutical
composition. General
principles of therapeutic formulations of cell compositions are found in Cell
Therapy: Stem Cell
Transplantation, Gene Therapy, and Cellular Immunotherapy, G. Morstyn & W.
Sheridan eds,
Cambridge University Press, 1996, and Hematopoietic Stem Cell Therapy, E.
Ball, J. Lister & P.
Law, Churchill Livingstone, 2000, the disclosures are incorporated herein by
reference.
1007151 Useful descriptions of neurons derived from stem cells and methods of
making thereof
can be found, for example, in Kirkeby et al., Cell Rep, 2012, 1:703-714; Kriks
et al., Nature,
2011, 480.547-551; Wang et al., Stem Cell Reports, 2018, 11(1).171-182, Lorenz
Studer,
-Chapter 8 - Strategies for Bringing Stem Cell-Derived Dopamine Neurons to the
clinic-The
NYSTEM Trial" in Progress in Brain Research, 2017, volume 230, pg. 191-212;
Liu et al., Nat
Protoc, 2013, 8:1670-1679; Upadhya et al., Curr Protoc Stem Cell Biol, 38,
2D.7.1-2D.7.47; US
Publication Appl. No. 20160115448, and US8,252,586; US8,273,570; US9,487,752
and
US10,093,897, the contents are incorporated herein by reference in their
entirety.
1007161 In addition to DA neurons, other neuronal cells, precursors, and
progenitors thereof can
be differentiated from the HIP cells outlined herein by culturing the cells in
medium comprising
one or more factors or additive. Non-limiting examples of factors and
additives include GDNF,
208
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, SMAD inhibitor, Wnt
antagonist,
SHH signaling activator, and any combination thereof. In some embodiments, the
SMAD
inhibitor is selected from the group consisting of SB431542, LDN-193189,
Noggin PD169316,
SB203580, LY364947, A77-01, A-83-01, BMP4, 6W788388, GW6604, SB-505124,
lerdelimumab, metelimumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276,
LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6,
NPC-
30345, K 26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-
1,
dorsomorphin dihydrochloride and derivatives thereof. In some embodiments, the
Wnt
antagonist is selected from the group consisting of XAV939, DKK1, DKK-2, DKK-
3, DKK-4,
SFRP-1, SFRP-2, SFRP-3, SFRP-4, SFRP-5, WIF-1, Soggy, IWP-2, IWR1, ICG-001,
KY0211,
Wnt-059, LGK974, IWP-L6 and derivatives thereof. In some embodiments, the SEIR
signaling
activator is selected from the group consisting of Smoothened agonist (SAG),
SAG analog, SHE,
C25-SHH, C24-SHH, purmorphamine, Hg-Ag and/or derivatives thereof.
1007171 In some embodiments, the neurons express one or more of the markers
selected from
the group consisting of glutamate ionotropic receptor NA/IDA type subunit 1
GRINE glutamate
decarboxylase 1 GAD1, gamma-aminobutyric acid GABA, tyrosine hydroxylase TH,
LEVI
homeobox transcription factor 1-alpha LMX1A, Forkhead box protein 01 FOX01,
Forkhead
box protein A2 FOXA2, Forkhead box protein 04 FOX04, F0XG1, 2',3'-cyclic-
nucleotide 3'-
phosphodiesterase CNP, myelin basic protein MBP, tubulin beta chain 3 TUB3,
tubulin beta
chain 3 NEUN, solute carrier family 1 member 6 SLC1A6, SST, PV, calbindin,
RAX, LHX6,
LHX8, DLX1, DLX2, DLX5, DLX6, SOX6, MAFB, NPAS1, ASCL1, SIX6, OLIG2, NKX2.1,
NKX2.2, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, ChAT, NGFI-B,
c-fos, CRF, RAX, POMC, hypocretin, NADPH, NGF, Ach, VAChT, PAX6, EMX2p75,
COR1N, TUJ1, NURR1, and/or any combination thereof.
7. Glial cells
1007181 In some embodiments, the neural cells described include glial cells
such as, but not
limited to, microglia, astrocytes, oligodendrocytes, ependymal cells and
Schwann cells, glial
precursors, and glial progenitors thereof are produced by differentiating
pluripotent stem cells
into therapeutically effective glial cells and the like. Differentiation of
hypoimmunogenic
209
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
pluripotent stem cells produces hypoimmunogenic neural cells, such as
hypoimmunogenic glial
cells.
1007191 In some embodiments, glial cells, precursors, and progenitors thereof
generated by
culturing pluripotent stem cells in medium comprising one or more agents
selected from the
group consisting of retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, a
TGFbeta
inhibitor, a BMP signaling inhibitor, a SEM signaling activator, FGF, platelet
derived growth
factor PDGF, PDGFR-alpha, HGF, IGF1, noggin, SHH, doisomoiphin, noggin, and
any
combination thereof. In certain instances, the BMP signaling inhibitor is
LDN193189,
SB431542, or a combination thereof. In some embodiments, the glial cells
express NKX2.2,
PAX6, SOX10, brain derived neurotrophic factor BDNF, neutrotrophin-3 NT-3, NT-
4, EGF,
ciliary neurotrophic factor CNTF, nerve growth factor NGF, FGF8, EGFR, OLIG1,
OLIG2,
myelin basic protein MBP, GAP-43, LNGFR, nestin, GFAP, CD11b, CD11 c, CX3CR1,
P2RY12, IBA-1, TMEM119, CD45, and any combination thereof. Exemplary
differentiation
medium can include any specific factors and/or small molecules that may
facilitate or enable the
generation of a glial cell type as recognized by those skilled in the art.
[00720] To determine if the cells generated according to the in vitro
differentiation protocol
display glial cell characteristics and features, the cells can be transplanted
into an animal model.
In some embodiments, the glial cells are injected into an immunocompromised
mouse, e.g., an
immunocompromised shiverer mouse. The glial cells are administered to the
brain of the mouse
and after a pre-selected amount of time the engrafted cells are evaluated. In
some instances, the
engrafted cells in the brain are visualized by using immunostaining and
imaging methods. In
some embodiments, it is determined that the glial cells express known glial
cell biomarkers.
[00721] Useful methods for generating glial cells, precursors, and progenitors
thereof from stem
cells are found, for example, in US7,579,188; US7,595,194; US8,263,402;
US8,206,699;
US8,252,586; US9,193,951; US9,862,925; US8,227,247; US9,709,553;
US2018/0187148;
US2017/0198255; US2017/0183627; US2017/0182097; US2017/253856; US2018/0236004;

W02017/172976; and W02018/093681. Methods for differentiating pluripotent stem
cells are
described in, e.g., Kikuchi et al., Nature, 2017, 548, 592-596; Kriks et al.,
Nature, 2011, 547-
551; Doi et al., Stem Cell Reports, 2014, 2, 337-50; Perrier et al., Proc Natl
Acad Sci USA,
2004, 101, 12543-12548; Chambers et al., Nat Biotechnol, 2009, 27, 275-280;
and Kirkeby et al.,
Cell Reports, 2012, 1, 703-714.
210
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007221 The efficacy of neural cell transplants for spinal cord injury can be
assessed in, for
example, a rat model for acutely injured spinal cord, as described by
McDonald, et al., Nat.
Med., 1999, 5:1410) and Kim, et al., Nature, 2002, 418:50. For instance,
successful transplants
may show transplant-derived cells present in the lesion 2-5 weeks later,
differentiated into
astrocytes, oligodendrocytes, and/or neurons, and migrating along the spinal
cord from the
lesioned end, and an improvement in gait, coordination, and weight-bearing.
Specific animal
models are selected based on the neural cell type and neurological disease or
condition to be
treated.
1007231 The neural cells can be administered in a manner that permits them to
engraft to the
intended tissue site and reconstitute or regenerate the functionally deficient
area. For instance,
neural cells can be transplanted directly into parenchymal or intrathecal
sites of the central
nervous system, according to the disease being treated. In some embodiments,
any of the neural
cells described herein including cerebral endothelial cells, neurons,
dopaminergic neurons,
ependymal cells, astrocytes, microglial cells, oligodendrocytes, and Schwann
cells are injected
into a patient by way of intravenous, intraspinal, intracerebroventricular,
intrathecal, intra-
arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, intra-
abdominal,
intraocular, retrobulbar and combinations thereof In some embodiments, the
cells are injected
or deposited in the form of a bolus injection or continuous infusion. In
certain embodiments, the
neural cells are administered by injection into the brain, apposite the brain,
and combinations
thereof. The injection can be made, for example, through a burr hole made in
the subject's skull.
Suitable sites for administration of the neural cell to the brain include, but
are not limited to, the
cerebral ventricle, lateral ventricles, cisterna magna, putamen, nucleus
basalis, hippocampus
cortex, striatum, caudate regions of the brain and combinations thereof.
1007241 Additional descriptions of neural cells including dopaminergic neurons
for use in the
present disclosure are found in W02020/018615, the disclosure is herein
incorporated by
reference in its entirety.
8. Endothelial Cells
1007251 Provided herein are hypoimmunogenic pluripotent cells that are
differentiated into
various endothelial cell types for subsequent transplantation or engraftment
into subjects (e.g.,
211
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
recipients). As will be appreciated by those in the art, the methods for
differentiation depend on
the desired cell type using known techniques
1007261 In some embodiments, the endothelial cells differentiated from the
subject
hypoimmunogenic pluripotent cells are administered to a patient, e.g., a human
patient in need
thereof. The endothelial cells can be administered to a patient suffering from
a disease or
condition such as, but not limited to, cardiovascular disease, vascular
disease, peripheral vascular
disease, ischemic disease, myocardial infarction, congestive heart failure,
peripheral vascular
obstructive disease, stroke, reperfusion injury, limb ischemia, neuropathy
(e.g., peripheral
neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney
failure, and the like),
diabetes, rheumatoid arthritis, osteoporosis, vascular injury, tissue injury,
hypertension, angina
pectoris and myocardial infarction due to coronary artery disease, renal
vascular hypertension,
renal failure due to renal artery stenosis, claudication of the lower
extremities, and the like. In
certain embodiments, the patient has suffered from or is suffering from a
transient ischemic
attack or stroke, which in some cases, may be due to cerebrovascular disease.
In some
embodiments, the engineered endothelial cells are administered to treat tissue
ischemia e.g., as
occurs in atherosclerosis, myocardial infarction, and limb ischemia and to
repair of injured blood
vessels. In some instances, the cells are used in bioengineering of grafts.
1007271 For instance, the endothelial cells can be used in cell therapy for
the repair of ischemic
tissues, formation of blood vessels and heart valves, engineering of
artificial vessels, repair of
damaged vessels, and inducing the formation of blood vessels in engineered
tissues (e.g., prior to
transplantation). Additionally, the endothelial cells can be further modified
to deliver agents to
target and treat tumors.
1007281 In certain embodiments, provided herein is a method of repair or
replacement for tissue
in need of vascular cells or vascularization. The method involves
administering to a human
patient in need of such treatment, a composition containing the isolated
endothelial cells to
promote vascularization in such tissue. The tissue in need of vascular cells
or vascularization can
be a cardiac tissue, liver tissue, pancreatic tissue, renal tissue, muscle
tissue, neural tissue, bone
tissue, among others, which can be a tissue damaged and characterized by
excess cell death, a
tissue at risk for damage, or an artificially engineered tissue.
1007291 In some embodiments, vascular diseases, which may be associated with
cardiac
diseases or disorders can be treated by administering endothelial cells, such
as but not limited to,
212
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
definitive vascular endothelial cells and endocardial endothelial cells
derived as described herein.
Such vascular diseases include, but are not limited to, coronary artery
disease, cerebrovascular
disease, aortic stenosis, aortic aneurysm, peripheral artery disease,
atherosclerosis, varicose
veins, angiopathy, infarcted area of heart lacking coronary perfusion, non-
healing wounds,
diabetic or non-diabetic ulcers, or any other disease or disorder in which it
is desirable to induce
formation of blood vessels.
1007301 In certain embodiments, the endothelial cells are used for improving
prosthetic implants
(e.g., vessels made of synthetic materials such as Dacron and Gortex.) which
are used in vascular
reconstructive surgery. For example, prosthetic arterial grafts are often used
to replace diseased
arteries which perfuse vital organs or limbs In other embodiments, the
engineered endothelial
cells are used to cover the surface of prosthetic heart valves to decrease the
risk of the formation
of emboli by making the valve surface less thrombogenic.
1007311 The endothelial cells outlined can be transplanted into the patient
using well known
surgical techniques for grafting tissue and/or isolated cells into a vessel.
In some embodiments,
the cells are introduced into the patient's heart tissue by injection (e.g.,
intramyocardial injection,
intracoronary injection, trans-endocardial injection, trans-cpicardial
injection, percutancous
injection), infusion, grafting, and implantation.
1007321 Administration (delivery) of the endothelial cells includes, but is
not limited to,
subcutaneous or parenteral including intravenous, intraarteri al (e.g.,
intracoronary),
intramuscular, intraperitoneal, intramyocardial, trans-endocardial, trans-
epicardial, intranasal
administration as well as intrathecal, and infusion techniques.
1007331 As will be appreciated by those in the art, the HIP derivatives are
transplanted using
techniques known in the art that depends on both the cell type and the
ultimate use of these cells.
In some embodiments, the cells differentiated from the subject HiPs provided
herein are
transplanted either intravenously or by injection at particular locations in
the patient. When
transplanted at particular locations, the cells may be suspended in a gel
matrix to prevent
dispersion while they take hold.
1007341 Exemplary endothelial cell types include, but are not limited to, a
capillary endothelial
cell, vascular endothelial cell, aortic endothelial cell, arterial endothelial
cell, venous endothelial
cell, renal endothelial cell, brain endothelial cell, liver endothelial cell,
and the like.
213
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007351 The endothelial cells outlined herein can express one or more
endothelial cell markers.
Non-limiting examples of such markers include VE-cadherin (CD 144), ACE
(angiotensin-
converting enzyme) (CD 143), BNH9/BNF13, CD31, CD34, CD54 (ICAM-1), CD62E (E-
Selectin), CD105 (Endoglin), CD146, Endocan (ESM-1), Endoglyx-1, Endomucin,
Eotaxin-3,
EPAS1 (Endothelial PAS domain protein 1), Factor VIII related antigen, FLI-1,
F1k-1 (KDR,
VEGFR-2), FLT-1 (VEGFR-1), GATA2, GBP-1 (guanylate- binding protein-1), GRO-
alpha,
HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, LYVE-1, MRB (magic
roundabout),
Nucleolin, PAL-E (pathologische anatomie Leiden- endothelium), RTKs, sVCAM-1,
TALI,
TEM1 (Tumor endothelial marker 1), TEM5 (Tumor endothelial marker 5), TEM7
(Tumor
endothelial marker 7), thrombomodulin (TM, CD141), VCAM-1 (vascular cell
adhesion
molecule- 1) (CD106), VEGF, vWF (von Willebrand factor), ZO-1, endothelial
cell-selective
adhesion molecule (ESAM), CD102, CD93, CD184, CD304, and DLL4.
1007361 In some embodiments, the endothelial cells are genetically modified to
express an
exogenous gene encoding a protein of interest such as but not limited to an
enzyme, hormone,
receptor, ligand, or drug that is useful for treating a disorder/condition or
ameliorating symptoms
of the disorder/condition. Standard methods for genetically modifying
endothelial cells are
described, e.g., in US5,674,722.
1007371 Such endothelial cells can be used to provide constitutive synthesis
and delivery of
polypeptides or proteins, which are useful in prevention or treatment of
disease In this way, the
polypeptide is secreted directly into the bloodstream or other area of the
body (e.g., central
nervous system) of the individual. In some embodiments, the endothelial cells
can be modified to
secrete insulin, a blood clotting factor (e.g., Factor VIII or von Willebrand
Factor), alpha-1
antitrypsin, adenosine deaminase, tissue plasminogen activator, interleukins
(e.g., IL-1, IL-2, IL-
3), and the like.
1007381 In certain embodiments, the endothelial cells can be modified in a way
that improves
their performance in the context of an implanted graft. Non-limiting
illustrative examples include
secretion or expression of a thrombolytic agent to prevent intraluminal clot
formation, secretion
of an inhibitor of smooth muscle proliferation to prevent luminal stenosis due
to smooth muscle
hypertrophy, and expression and/or secretion of an endothelial cell mitogen or
autocrine factor to
stimulate endothelial cell proliferation and improve the extent or duration of
the endothelial cell
lining of the graft lumen
214
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007391 In some embodiments, the engineered endothelial cells are utilized for
delivery of
therapeutic levels of a secreted product to a specific organ or limb. For
example, a vascular
implant lined with endothelial cells engineered (transduced) in vitro can be
grafted into a specific
organ or limb. The secreted product of the transduced endothelial cells will
be delivered in high
concentrations to the perfused tissue, thereby achieving a desired effect to a
targeted anatomical
location.
1007401 In oilier embodiments, the endothelial cells are genetically modified
to contain a gene
that disrupts or inhibits angiogenesis when expressed by endothelial cells in
a vascularizing
tumor. In some cases, the endothelial cells can also be genetically modified
to express any one of
the selectable suicide genes described herein which allows for negative
selection of grafted
endothelial cells upon completion of tumor treatment.
1007411 In some embodiments, endothelial cells described herein are
administered to a recipient
subject to treat a vascular disorder selected from the group consisting of
vascular injury,
cardiovascular disease, vascular disease, peripheral vascular disease,
ischemic disease,
myocardial infarction, congestive heart failure, peripheral vascular
obstructive disease,
hypertension, ischemic tissue injury, reperfusion injury, limb ischemia,
stroke, neuropathy (e.g.,
peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver
failure, kidney failure,
and the like), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular
disease, hypertension,
angina pectoris and myocardial infarction due to coronary artery disease,
renal vascular
hypertension, renal failure due to renal artery stenosis, claudication of the
lower extremities,
other vascular condition or disease.
1007421 In some embodiments, the hypoimmunogenic pluripotent cells are
differentiated into
endothelial colony forming cells (ECFCs) to form new blood vessels to address
peripheral
arterial disease. Techniques to differentiate endothelial cells are known.
See, e.g., Prasain et al.,
doi: 10.1038/nbt.3048, incorporated herein by reference in its entirety and
specifically for the
methods and reagents for the generation of endothelial cells from human
pluripotent stem cells,
and also for transplantation techniques. Differentiation can be assayed as is
known in the art,
generally by evaluating the presence of endothelial cell associated or
specific markers or by
measuring functionally.
1007431 In some embodiments, the method of producing a population of
hypoimmunogenic
endothelial cells from a population of hypoimmunogenic induced pluripotent
stem (HIP) cells by
215
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
in vitro differentiation comprises: (a) culturing a population of HIP cells in
a first culture
medium comprising a GSK inhibitor; (b) culturing the population of HIP cells
in a second
culture medium comprising VEGF and bFGF to produce a population of pre-
endothelial cells;
and (c) culturing the population of pre-endothelial cells in a third culture
medium comprising a
ROCK inhibitor and an ALK inhibitor to produce a population of hypoimmunogenic
endothelial
cells.
1007441 In some embodiments, the GSK inhibitor is CHIR-99021, a derivative
thereof, or a
variant thereof. In some instances, the GSK inhibitor is at a concentration
ranging from about 1
mM to about 10 mM. In some embodiments, the ROCK inhibitor is Y-27632, a
derivative
thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a
concentration ranging
from about 1 pM to about 20 pM. In some embodiments, the ALK inhibitor is SB-
431542, a
derivative thereof, or a variant thereof. In some instances, the ALK inhibitor
is at a concentration
ranging from about 0.5 pM to about 10 pM.
1007451 In some embodiments, the first culture medium comprises from 2 pM to
about 10 pM
of CHIR-9902 L In some embodiments, the second culture medium comprises 50
ng/ml VEGF
and 10 ng/ml bFGF. In other embodiments, the second culture medium further
comprises Y-
27632 and SB-431542. In various embodiments, the third culture medium
comprises 10 pM Y-
27632 and 1 pM SB-431542. In certain embodiments, the third culture medium
further
comprises VEGF and bFGF. In particular instances, the first culture medium
and/or the second
medium is absent of insulin.
1007461 The cells provided herein can be cultured on a surface, such as a
synthetic surface to
support and/or promote differentiation of hypoimmunogenic pluripotent cells
into cardiac cells.
In some embodiments, the surface comprises a polymer material including, but
not limited to, a
homopolymer or copolymer of selected one or more acrylate monomers. Non-
limiting examples
of acrylate monomers and methacrylate monomers include tetra(ethylene glycol)
diacrylate,
glycerol dimethacrylate, 1,4-butanediol dimethacrylate, poly(ethylene glycol)
diacrylate,
di(ethylene glycol) dimethacrylate, tetra(ethyiene glycol) dimethacrylate, 1,6-
hexanediol
propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane
benzoate diacrylate,
trimethylolpropane eihoxylate (1 EO/QH) methyl, tricyclo[5.2.1.02,6] decane
dimethanol
diacrylate, neopentyl glycol exhoxylate diacrylate, and trimethylolpropane
triacrylate. Acrylate
216
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
synthesized as known in the art or obtained from a commercial vendor, such as
Polysciences,
Inc., Sigma Aldrich, Inc. and Sartomer, Inc.
1007471 In some embodiments, the endothelial cells may be seeded onto a
polymer matrix. In
some cases, the polymer matrix is biodegradable. Suitable biodegradable
matrices are well
known in the art and include collagen-GAG, collagen, fibrin, PLA, PGA, and
PLA/PGA co-
polymers. Additional biodegradable materials include poly(anhydrides),
poly(hydroxy acids),
poly(ortho esters), poly(propylfumerates), poly(caprolactones), polyamides,
polyamino acids,
polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes and

polysaccharides.
1007481 Non-biodegradable polymers may also be used as well. Other non-
biodegradable, yet
biocompatible polymers include polypyrrole, polyanibnes, polythiophene,
polystyrene,
polyesters, non-biodegradable polyurethanes, polyureas, poly(ethylene vinyl
acetate),
polypropylene, polymethacrylate, polyethylene, polycarbonates, and
poly(ethylene oxide). The
polymer matrix may be formed in any shape, for example, as particles, a
sponge, a tube, a
sphere, a strand, a coiled strand, a capillary network, a film, a fiber, a
mesh, or a sheet. The
polymer matrix can be modified to include natural or synthetic extracellular
matrix materials and
factors.
1007491 The polymeric material can be dispersed on the surface of a support
material. Useful
support materials suitable for culturing cells include a ceramic substance, a
glass, a plastic, a
polymer or co-polymer, any combinations thereof, or a coating of one material
on another. In
some instances, a glass includes soda-lime glass, pyrex glass, vycor glass,
quartz glass, silicon,
or derivatives of these or the like.
1007501 In some instances, plastics or polymers including dendritic polymers
include poly(vinyl
chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-
maleic anhydride),
poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon
polymers,
polystyrenes, polypropylene, polyethyleneimine or derivatives of these or the
like. In some
instances, copolymers include poly(vinyl acetate-co-maleic anhydride),
poly(styrene-co-maleic
anhydride), poly(ethylene-co-acrylic acid) or derivatives of these or the
like.
1007511 In some embodiments, the population of hypoimmunogenic endothelial
cells is isolated
from non-endothelial cells. In some embodiments, the isolated population of
hypoimmunogenic
endothelial cells are expanded prior to administration. In certain
embodiments, the isolated
217
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
population of hypoimmunogenic endothelial cells are expanded and cryopreserved
prior to
administration.
[00752] Additional descriptions of endothelial cells for use in the methods
provided herein are
found in W02020/018615, the disclosure is herein incorporated by reference in
its entirety.
9. Thyroid Cells
[00753] In some embodiments, the hypoimmunogenic pluripotent cells are
differentiated into
thyroid progenitor cells and thyroid follicular organoids that can secrete
thyroid hormones to
address autoimmune thyroiditis. Techniques to differentiate thyroid cells are
known the art. See,
e.g., Kurmann et al., Cell Stem Cell, 2015 Nov 5,17(5).527-42, incorporated
herein by reference
in its entirety and specifically for the methods and reagents for the
generation of thyroid cells
from human pluripotent stem cells, and also for transplantation techniques.
Differentiation can be
assayed as is known in the art, generally by evaluating the presence of
thyroid cell associated or
specific markers or by measuring functionally.
10. Hepatocytes
1007541 In some embodiments, the hypoimmunogenic induced pluripotent stem
(HIP) cells are
differentiated into hepatocytes to address loss of the hepatocyte functioning
or cirrhosis of the
liver. There are a number of techniques that can be used to differentiate HIP
cells into
hepatocytes; see for example, Pettinato et al., doi: 10.1038/spre32888,
Snykers et al., Methods
Mol Biol, 2011 698:305-314, Si-Tayeb et al., Hepatology, 2010, 51:297-305 and
Asgari et al,
Stem Cell Rev, 2013, 9(4):493- 504, all of which are incorporated herein by
reference in their
entirety and specifically for the methodologies and reagents for
differentiation. Differentiation
can be assayed as is known in the art, generally by evaluating the presence of
hepatocyte
associated and/or specific markers, including, but not limited to, albumin,
alpha fetoprotein, and
fibrinogen. Differentiation can also be measured functionally, such as the
metabolization of
ammonia, LDL storage and uptake, ICG uptake and release, and glycogen storage.
11. Pancreatic Islet Cells
[00755] In some embodiments, pancreatic islet cells (also referred to as
pancreatic beta cells)
are derived from the hypoimmunogenic induced pluripotent stem (HIP) cells
described herein. In
some instances, hypoimmunogenic pluripotent cells that are differentiated into
various pancreatic
218
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
islet cell types are transplanted or engrafted into subjects (e.g.,
recipients). As will be
appreciated by those in the art, the methods for differentiation depend on the
desired cell type
using known techniques. Exemplary pancreatic islet cell types include, but are
not limited to,
pancreatic islet progenitor cell, immature pancreatic islet cell, mature
pancreatic islet cell, and
the like. In some embodiments, pancreatic cells described herein are
administered to a subject to
treat diabetes.
1007561 In some embodiments, pancreatic islet cells are derived from the
hypoimmunogenic
pluripotent cells described herein. Useful method for differentiating
pluripotent stem cells into
pancreatic islet cells are described, for example, in US9,683,215;
US9,157,062; and
US8,927,280.
1007571 In some embodiments, the pancreatic islet cells produced by the
methods as disclosed
herein secretes insulin. In some embodiments, a pancreatic islet cell exhibits
at least two
characteristics of an endogenous pancreatic islet cell, for example, but not
limited to, secretion of
insulin in response to glucose, and expression of beta cell markers.
1007581 Exemplary beta cell markers or beta cell progenitor markers include,
but are not limited
to, c-peptide, Pdxl, glucose transporter 2 (Glut2), HNF6, VEGF, glucokinasc
(GCK),
prohormone convertase (PC 1/3), Cdcpl, NeuroD, Ngn3, Nkx2.2, Nkx6.1, Nkx6.2,
Pax4, Pax6,
Ptfla, Isll, Sox9, Sox17, and FoxA2.
1007591 In some embodiments, the isolated pancreatic islet cells produce
insulin in response to
an increase in glucose. In various embodiments, the isolated pancreatic islet
cells secrete insulin
in response to an increase in glucose. In some embodiments, the cells have a
distinct morphology
such as a cobblestone cell morphology and/or a diameter of about 17 pm to
about 25 pm.
1007601 In some embodiments, the hypoimmunogenic pluripotent cells are
differentiated into
beta-like cells or islet organoids for transplantation to address type I
diabetes mellitus (T1DM).
Cell systems are a promising way to address T1DM, see, e.g., Ellis et al, Nat
Rev Gastroenterol
Hepatol. 2017 Oct;14(10):612-628, incorporated herein by reference.
Additionally, Pagliuca et
al. (Cell, 2014, 159(2):428-39) reports on the successful differentiation of
I3-cells from human
iPSCs, the contents incorporated herein by reference in its entirety and in
particular for the
methods and reagents outlined there for the large-scale production of
functional human 13 cells
from human pluripotent stem cells). Furthermore, Vegas et al. shows the
production of human 13
cells from human pluripotent stem cells followed by encapsulation to avoid
immune rejection by
219
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
the host; Vegas et al., Nat Med, 2016, 22(3):306-11, incorporated herein by
reference in its
entirety and in particular for the methods and reagents outlined there for the
large-scale
production of functional human 13 cells from human pluripotent stem cells.
1007611 In some embodiments, the method of producing a population of
hypoimmunogenic
pancreatic islet cells from a population of hypoimmunogenic induced
pluripotent stem (HIP)
cells by in vitro differentiation comprises: (a) culturing the population of
HIP cells in a first
culture medium comprising one or more factors selected from the group
consisting insulin-like
growth factor, transforming growth factor, FGF, EGF, HGF, SHH, VEGF,
transforming growth
factor-b superfamily, BMP2, BMP7, a GSK inhibitor, an ALK inhibitor, a BMP
type 1 receptor
inhibitor, and retinoic acid to produce a population of immature pancreatic
islet cells; and (b)
culturing the population of immature pancreatic islet cells in a second
culture medium that is
different than the first culture medium to produce a population of hypoimmune
pancreatic islet
cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative
thereof, or a variant
thereof. In some instances, the GSK inhibitor is at a concentration ranging
from about 2 mM to
about 10 mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative
thereof, or a
variant thereof. In some instances, the ALK inhibitor is at a concentration
ranging from about 1
pM to about 10 pM. In some embodiments, the first culture medium and/or second
culture
medium are absent of animal serum.
1007621 In some embodiments, the population of hypoimmunogenic pancreatic
islet cells is
isolated from non-pancreatic islet cells. In some embodiments, the isolated
population of
hypoimmunogenic pancreatic islet cells are expanded prior to administration.
In certain
embodiments, the isolated population of hypoimmunogenic pancreatic islet cells
are expanded
and cryopreserved prior to administration.
1007631 Differentiation is assayed as is known in the art, generally by
evaluating the presence of
13 cell associated or specific markers, including but not limited to, insulin.
Differentiation can
also be measured functionally, such as measuring glucose metabolism, see
generally Muraro et
al., Cell Syst. 2016 Oct 26; 3(4): 385-394.e3, hereby incorporated by
reference in its entirety,
and specifically for the biomarkers outlined there. Once the beta cells are
generated, they can be
transplanted (either as a cell suspension or within a gel matrix as discussed
herein) into the portal
vein/liver, the omentum, the gastrointestinal mucosa, the bone marrow, a
muscle, or
subcutaneous pouches.
220
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007641 Additional descriptions of pancreatic islet cells including
dopaminergic neurons for use
in the present disclosure are found in W02020/018615, the disclosure is herein
incorporated by
reference in its entirety.
12. Retinal Pigmented Epithelium (RPE) Cells
1007651 Provided herein are retinal pigmented epithelium (RPE) cells derived
from the
hypoimmunogenic induced pluripotent stem (HIP) cells described. For instance,
human RPE
cells can be produced by differentiating human HIP cells. In some embodiments,

hypoimmunogenic pluripotent cells that are differentiated into various RPE
cell types are
transplanted or engrafted into subjects (e.g., recipients). As will be
appreciated by those in the
art, the methods for differentiation depend on the desired cell type using
known techniques.
1007661 The term "RPE" cells refers to pigmented retinal epithelial cells
having a genetic
expression profile similar or substantially similar to that of native RPE
cells. Such RPE cells
derived from pluripotent stem cells may possess the polygonal, planar sheet
morphology of
native RPE cells when grown to confluence on a planar substrate.
1007671 The RPE cells can be implanted into a patient suffering from macular
degeneration or a
patient having damaged RPE cells. In some embodiments, the patient has age-
related macular
degeneration (AMD), early AMD, intermediate AMD, late AMD, non-neovascular age-
related
macular degeneration, dry macular degeneration (dry age-related macular
degeneration), wet
macular degeneration (wet age-real ted macular degeneration), juvenile macular
degeneration
(JMD) (e.g., Stargardt disease, Best disease, and juvenile retinoschisis),
Leber's Congenital
Ameurosis, or retinitis pigmentosa. In other embodiments, the patient suffers
from retinal
detachment.
1007681 Exemplary RPE cell types include, but are not limited to, retinal
pigmented epithelium
(RPE) cell, RPE progenitor cell, immature RPE cell, mature RPE cell,
functional RPE cell, and
the like.
1007691 Useful methods for differentiating pluripotent stem cells into RPE
cells are described
in, for example, US9,458,428 and US9,850,463, the disclosures are herein
incorporated by
reference in their entirety, including the specifications. Additional methods
for producing RPE
cells from human induced pluripotent stem cells can be found in, for example,
Lamba et al.,
PNAS, 2006, 103(34): 12769-12774; Mellough et al, Stem Cells, 2012, 30(4):673-
686; Idelson
221
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
eta!, Cell Stem Cell, 2009, 5(4): 396-408; Rowland et al, Journal of Cellular
Physiology, 2012,
227(2):457-466, Buchholz et al, Stem Cells Trans Med, 2013, 2(5): 384-393, and
da Cruz et al,
Nat Biotech, 2018, 36:328-337.
1007701 Human pluripotent stem cells have been differentiated into RPE cells
using the
techniques outlined in Kamao et al, Stem Cell Reports 2014:2:205-18, hereby
incorporated by
reference in its entirety and in particular for the methods and reagents
outlined there for the
differentiation techniques and reagents, see also Mandai et al., N Engl J Med,
2017, 376.1038-
1046, the contents herein incorporated in its entirety for techniques for
generating sheets of RPE
cells and transplantation into patients. Differentiation can be assayed as is
known in the art,
generally by evaluating the presence of RPE associated and/or specific markers
or by measuring
functionally. See for example Kamao et al., Stem Cell Reports, 2014, 2(2):205-
18, the contents
incorporated herein by reference in its entirety and specifically for the
markers outlined in the
first paragraph of the results section.
1007711 In some embodiments, the method of producing a population of
hypoimmunogenic
retinal pigmented epithelium (RPE) cells from a population of hypoimmunogenic
pluripotent
cells by in vitro differentiation comprises: (a) culturing the population of
hypoimmunogenic
pluripotent cells in a first culture medium comprising any one of the factors
selected from the
group consisting of activin A, bFGF, BMP4/7, DKK1, IGF1, noggin, a BMP
inhibitor, an ALK
inhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce a population of
pre-RPE cells;
and (b) culturing the population of pre-RPE cells in a second culture medium
that is different
than the first culture medium to produce a population of hypoimmunogenic RPE
cells. In some
embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a
variant thereof. In some
instances, the ALK inhibitor is at a concentration ranging from about 2 mM to
about 10 pM. In
some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a
variant thereof In
some instances, the ROCK inhibitor is at a concentration ranging from about I
pM to about 10
pM. In some embodiments, the first culture medium and/or second culture medium
are absent of
animal serum.
1007721 Differentiation can be assayed as is known in the art, generally by
evaluating the
presence of RPE associated and/or specific markers or by measuring
functionally. See for
example Kamao et al., Stem Cell Reports, 2014, 2(2):205-18, the contents are
herein
incorporated by reference in its entirety and specifically for the results
section.
222
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007731 Additional descriptions of RPE cells for use in the present disclosure
are found in
W02020/018615, the disclosure is herein incorporated by reference in its
entirety.
1007741 For therapeutic application, cells prepared according to the disclosed
methods can
typically be supplied in the form of a pharmaceutical composition comprising
an isotonic
excipient, and are prepared under conditions that are sufficiently sterile for
human
administration. For general principles in medicinal formulation of cell
compositions, see "Cell
Therapy. Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy,"
by Morstyn
& Sheridan eds, Cambridge University Press, 1996; and "Hematopoietic Stem Cell
Therapy," E.
D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The cells can be
packaged in a device
or container suitable for distribution or clinical use.
13. T Lymphocytes
1007751 Provided herein, T lymphocytes (T cells) are derived from the
hypoimmunogenic
induced pluripotent stem (HIP) cells described. Methods for generating T
cells, including CAR-T
cells, from pluripotent stem cells (e.g., iPSCs) are described, for example,
in Iriguchi et al.,
Nature Communications 12, 430 (2021); Themeli et al., Cell Stem Cell,
16(4):357-366 (2015);
Themeli et al., Nature Biotechnology 31:928-933 (2013).
1007761 In some embodiments, the hypoimmunogenic induced pluripotent stem cell-
derived T
cell includes a chimeric antigen receptor (CAR). Any suitable CAR can be
included in the
hypoimmunogenic induced pluripotent stem cell-derived T cell, including the
CARs described
herein. In some embodiments, the hypoimmunogenic induced pluripotent stem cell-
derived T
cell includes a polynucleotide encoding a CAR, wherein the polynucleotide is
inserted in a
genomic locus. In some embodiments, the polynucleotide is inserted into a safe
harbor or target
locus. In some embodiments, the polynucleotide is inserted in a B2M, CIITA,
TRAC, TRB, PD-
1 or CTLA-4 gene. Any suitable method can be used to insert the CAR into the
genomic locus
of the hypoimmunogenic cell including the gene editing methods described
herein (e.g., a
CRISPR/Cas system).
1007771 HIP-derived T cells provided herein are useful for the treatment of
suitable cancers
including, but not limited to, B cell acute lymphoblastic leukemia (B-ALL),
diffuse large B-cell
lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer,
colorectal cancer, lung
cancer, non-small cell lung cancer, acute myeloid lymphoid leukemia, multiple
myeloma, gastric
223
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma,
neuroblastoma, lung
squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.
U. Methods of Genetic Modifications
1007781 In some embodiments, a vector herein is a nucleic acid molecule
capable transferring
or transporting another nucleic acid molecule, including into the cell or into
genome of a cell.
The transferred nucleic acid is generally linked to, e.g., inserted into, the
vector nucleic acid
molecule. A vector may include sequences that direct autonomous replication in
a cell or may
include sequences sufficient to allow integration into host cell DNA. Useful
vectors include, for
example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids,
bacterial
artificial chromosomes, and viral vectors. Useful viral vectors include, e.g.,
replication defective
retroviruses and lentiviruses. Non-viral vectors may require a delivery
vehicle to facilitate entry
of the nucleic acid molecule into a cell.
1007791 A viral vector can comprise a nucleic acid molecule that includes
virus-derived nucleic
acid elements that typically facilitate transfer of the nucleic acid molecule
or integration into the
genome of a cell or to a viral particle that mediates nucleic acid transfer.
Viral particles will
typically include various viral components and sometimes also host cell
components in addition
to nucleic acid(s). A viral vector can comprise, e.g., a virus or viral
particle capable of
transferring a nucleic acid into a cell, or to the transferred nucleic acid
(e.g., as naked DNA).
Viral vectors and transfer plasmids can comprise structural and/or functional
genetic elements
that are primarily derived from a virus. A retroviral vector can comprise a
viral vector or
plasmid containing structural and functional genetic elements, or portions
thereof, that are
primarily derived from a retrovirus.
1007801 In some vectors described herein, at least part of one or more protein
coding regions
that contribute to or are essential for replication may be absent compared to
the corresponding
wild-type virus. This makes the viral vector replication-defective. In some
embodiments, the
vector is capable of transducing a target non-dividing host cell and/or
integrating its genome
into a host genome.
1007811 In some embodiments, the retroviral nucleic acid comprises one or more
of (e.g., all
of): a 5' promoter (e.g., to control expression of the entire packaged RNA), a
5' LTR (e.g., that
224
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
includes R (polyadenylation tail signal) and/or U5 which includes a primer
activation signal), a
primer binding site, a psi packaging signal, a RRE element for nuclear export,
a promoter
directly upstream of the transgene to control transgene expression, a
transgene (or other
exogenous agent element), a polypurine tract, and a 3' LTR (e.g., that
includes a mutated U3, a
R, and U5). In some embodiments, the retroviral nucleic acid further comprises
one or more of
a cPPT, a WPRE, and/or an insulator element.
[00782] A retrovirus typically replicates by reverse transcription of its
genomic RNA into a
linear double-stranded DNA copy and subsequently covalently integrates its
genomic DNA into
a host genome. The structure of a wild-type retrovirus genome often comprises
a 5' long
terminal repeat (LTR) and a 3' LTR, between or within which are located a
packaging signal to
enable the genome to be packaged, a primer binding site, integration sites to
enable integration
into a host cell genome and gag, pol and env genes encoding the packaging
components which
promote the assembly of viral particles More complex retroviruses have
additional features,
such as rev and RRE sequences in HIV, which enable the efficient export of RNA
transcripts of
the integrated provirus from the nucleus to the cytoplasm of an infected
target cell. In the
provirus, the viral genes are flanked at both ends by regions called long
terminal repeats
(LTRs). The LTRs are involved in proviral integration and transcription. LTRs
also serve as
enhancer-promoter sequences and can control the expression of the viral genes.
Encapsidation
of the retroviral RNAs occurs by virtue of a psi sequence located at the 5'
end of the viral
genome.
[00783] The LTRs themselves are typically similar (e.g., identical) sequences
that can be
divided into three elements, which are called U3, R and U5. U3 is derived from
the sequence
unique to the 3 end of the RNA. R is derived from a sequence repeated at both
ends of the RNA
and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes
of the three
elements can vary considerably among different retroviruses.
[00784] For the viral genome, the site of transcription initiation is
typically at the boundary
between U3 and R in one LTR and the site of poly (A) addition (termination) is
at the boundary
between R and U5 in the other LTR. U3 contains most of the transcriptional
control elements of
the provirus, which include the promoter and multiple enhancer sequences
responsive to cellular
and in some cases, viral transcriptional activator proteins. Some retroviruses
comprise any one
225
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
or more of the following genes that code for proteins that are involved in the
regulation of gene
expression: tot, rev, tax and rex.
1007851 With regard to the structural genes gag, pol and env themselves, gag
encodes the
internal structural protein of the virus. Gag protein is proteolytically
processed into the mature
proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes
the reverse
transcriptase (RT), which contains DNA polymerase, associated RNase H and
integrase (IN),
which mediate replication of the genome. The env gene encodes the surface (SU)
glycoprotein
and the transmembrane (TM) protein of the virion, which form a complex that
interacts
specifically with cellular receptor proteins. This interaction promotes
infection, e.g., by fusion
of the viral membrane with the cell membrane.
1007861 In a replication-defective retroviral vector genome gag, pol and env
may be absent or
not functional. The R regions at both ends of the RNA are typically repeated
sequences. U5 and
U3 represent unique sequences at the 5' and 3' ends of the RNA genome
respectively.
Retroviruses may also contain additional genes which code for proteins other
than gag, pol and
env. Examples of additional genes include (in HIV), one or more of vif, vpr,
vpx, vpu, tat, rev
and nef. EIAV has (amongst others) the additional gene S2.
1007871 Illustrative retroviruses suitable for use in particular embodiments,
include, but are not
limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma
virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus

(MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLY),
spumavirus,
Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma
Virus
(RSV)) and lentivirus.
1007881 In some embodiments the retrovirus is a Gammretrovirus. In some
embodiments the
retrovirus is an Epsilonretrovii us. In some embodiments the retrovirus is an
Alpharetrovirus. In
some embodiments the retrovirus is a Betaretro virus. In some embodiments the
retrovirus is a
Deltaretro virus. In some embodiments the retrovirus is a Spumaretrovirus. In
some
embodiments the retrovirus is an endogenous retrovirus. In some embodiments
the retrovirus is
alentivirus.
226
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1007891 In some embodiments, a retroviral or lentivirus vector further
comprises one or more
insulator elements, e.g., an insulator element described herein. In various
embodiments, the
vectors comprise a promoter operably linked to a polynucleotide encoding an
exogenous agent.
The vectors may have one or more LTRs, wherein either LTR comprises one or
more
modifications, such as one or more nucleotide substitutions, additions, or
deletions. The vectors
may further comprise one of more accessory elements to increase transduction
efficiency (e.g., a
cPPT/FLAP), viral packaging (e.g., a Psi (Y) packaging signal, RRE), and/or
other elements
that increase exogenous gene expression (e.g., poly (A) sequences), and may
optionally
comprise a WPRE or HPRE. In some embodiments, a lentiviral nucleic acid
comprises one or
more of, e.g., all of, e.g., from 5' to 3', a promoter (e.g., CMV), an R
sequence (e.g., comprising
TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse
transcription), a
DIS sequence (e.g., for genome dimerization), a psi packaging signal, a
partial gag sequence, an
RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear
import), a promoter
to drive expression of the exogenous agent, a gene encoding the exogenous
agent, a WPRE
sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for
reverse
transcription), an R sequence (e.g., for polyadenylation and termination), and
a U5 signal (e.g.,
for integration).
1007901 Illustrative lentiviruses include, but are not limited to: HIV (human
immunodeficiency
virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus;
the caprine
arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV);
feline
immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian

immunodeficiency virus (Sly). In some embodiments, HIV based vector backbones
(i.e., HIV
cis-acting sequence elements) are used.A lentivirus vector can comprise a
viral vector or
plasmid containing structural and functional genetic elements, or portions
thereof, including
Uflts that are primarily derived from a lentivirus.
1007911 In embodiments, a lentivirus vector (e.g., lentiviral expression
vector) may comprise a
lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral
particle. With respect
to elements such as cloning sites, promoters, regulatory elements,
heterologous nucleic
acids,etc., it is to be understood that the sequences of these elements can be
present in RNA
form in lentiviral particles and can be present in DNA form in DNA plasmids.
227
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00792] In embodiments, a lentivirus vector is a vector with sufficient
retroviral genetic
information to allow packaging of an RNA genome, in the presence of packaging
components,
into a viral particle capable of infecting a target cell. Infection of the
target cell can comprise
reverse transcription and integration into the target cell genome. The RLV
typically carries non-
viral coding sequences which are to be delivered by the vector to the target
cell. In
embodiments, an RLV is incapable of independent replication to produce
infectious retroviral
particles within the target cell. Usually the RLV lacks a functional gag-pol
and/or env gene
and/or other genes involved in replication. The vector may be configured as a
split-intron
vector, e.g., as described in PCT patent application WO 99/15683, which is
herein incorporated
by reference in its entirety.
[00793] In some embodiments, the lentivirus vector comprises a minimal viral
genome, e.g.,
the viral vector has been manipulated so as to remove the non-essential
elements and to retain
the essential elements in order to provide the required functionality to
infect, transduce and
deliver a nucleotide sequence of interest to a target host cell, e.g., as
described in WO 98/17815,
which is herein incorporated by reference in its entirety.
[00794] A minimal lentiviral genome may comprise, e.g., (5')R-U5-one or more
first nucleotide
sequences-U3-R(3')= However, the plasmid vector used to produce the lentiviral
genome within
a source cell can also include transcriptional regulatory control sequences
operably linked to the
lentiviral genome to direct transcription of the genome in a source cell.
These regulatory
sequences may comprise the natural sequences associated with the transcribed
retroviral
sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter
such as another
viral promoter, for example the CMV promoter. Some lentiviral genomes comprise
additional
sequences to promote efficient virus production. For example, in the case of
HIV, rev and RRE
sequences may be included.
[00795] In some embodiments, the rare-cutting endonuclease is introduced into
a cell containing
the target polynucleotide sequence in the form of a nucleic acid encoding a
rare-cutting
endonuclease. The process of introducing the nucleic acids into cells can be
achieved by any
suitable technique. Suitable techniques include calcium phosphate or lipid-
mediated transfection,
electroporation, and transduction or infection using a viral vector. In some
embodiments, the
nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a
modified
228
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
DNA, as described herein. In some embodiments, the nucleic acid comprises
mRNA. In some
embodiments, the nucleic acid comprises a modified mRNA, as described herein
(e.g., a
synthetic, modified mRNA).
1007961 The present disclosure contemplates altering target polynucleotide
sequences in any
manner which is available to the skilled artisan utilizing a gene editing
system (e.g.
CRISPR/Cas) of the present disclosure. Any CRISPR/Cas system that is capable
of altering a
target polynucleotide sequence in a cell can be used. Such CRISPR-Cas systems
can employ a
variety of Cas proteins (Haft et al. PLoS Comput Biol. 2005; 1(6)e60). The
molecular machinery
of such Cas proteins that allows the CRISPR/Cas system to alter target
polynucleotide sequences
in cells include RNA binding proteins, endo- and exo-nucleases, helicases, and
polymerases. In
some embodiments, the CRISPR/Cas system is a CRISPR Type I system. In some
embodiments,
the CRISPR/Cas system is a CRISPR Type II system. In some embodiments, the
CRISPR/Cas
system is a CRISPR Type V system.
1007971 The CRISPR/Cas systems of the present disclosure can be used to alter
any target
polynucleotide sequence in a cell. Those skilled in the art will readily
appreciate that desirable
target polynucleotide sequences to be altered in any particular cell may
correspond to any
genomic sequence for which expression of the genomic sequence is associated
with a disorder or
otherwise facilitates entry of a pathogen into the cell. For example, a
desirable target
polynucleotide sequence to alter in a cell may be a polynucleotide sequence
corresponding to a
genomic sequence which contains a disease associated single polynucleotide
polymorphism. In
such example, the CRISPR/Cas systems of the present disclosure can be used to
correct the
disease associated SNP in a cell by replacing it with a wild-type allele. As
another example, a
polynucleotide sequence of a target gene which is responsible for entry or
proliferation of a
pathogen into a cell may be a suitable target for deletion or insertion to
disrupt the function of the
target gene to prevent the pathogen from entering the cell or proliferating
inside the cell.
1007981 In some embodiments, the target polynucleotide sequence is a genomic
sequence. In
some embodiments, the target polynucleotide sequence is a human genomic
sequence. In some
embodiments, the target polynucleotide sequence is a mammalian genomic
sequence. In some
embodiments, the target polynucleotide sequence is a vertebrate genomic
sequence.
1007991 In some embodiments, a CRISPR/Cas system of the present disclosure
includes a Cas
protein and at least one to two ribonucleic acids that are capable of
directing the Cas protein to
229
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
and hybridizing to a target motif of a target polynucleotide sequence. As used
herein, "protein"
and "polypeptide" are used interchangeably to refer to a series of amino acid
residues joined by
peptide bonds (i.e., a polymer of amino acids) and include modified amino
acids (e.g.,
phosphorylated, glycated, glycosylated, etc.) and amino acid analogs.
Exemplary polypeptides or
proteins include gene products, naturally occurring proteins, homologs,
paralogs, fragments and
other equivalents, variants, and analogs of the above.
[00800] In some embodiments, a Cas protein comprises one or more amino acid
substitutions or
modifications. In some embodiments, the one or more amino acid substitutions
comprises a
conservative amino acid substitution. In some instances, substitutions and/or
modifications can
prevent or reduce proteolytic degradation and/or extend the half-life of the
polypeptide in a cell.
In some embodiments, the Cas protein can comprise a peptide bond replacement
(e.g., urea,
thiourea, carbamate, sulfonyl urea, etc.). In some embodiments, the Cas
protein can comprise a
naturally occurring amino acid. In some embodiments, the Cas protein can
comprise an
alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine,
phosphoserine,
etc.). In some embodiments, a Cas protein can comprise a modification to
include a moiety (e.g.,
PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).
[00801] [0007] In some embodiments, a Cas protein comprises a core
Cas protein,
isoform thereof, or any Cas-like protein with similar function or activity of
any Cas protein or
isoform thereof. In some embodiments, a Cas protein comprises a core Cas
protein. Exemplary
Cas core proteins include, but are not limited to Casl, Cas2, Cas3, Cas4,
Cas5, Cas6, Cas7, Cas8
and Cas9. In some embodiments, a Cas protein comprises type V Cas protein. In
some
embodiments, a Cas protein comprises a Cas protein of an E. coli subtype (also
known as
CASS2). Exemplary Cas proteins of the E. Coli subtype include, but are not
limited to Csel,
Cse2, Cse3, Cse4, and Cas5e. In some embodiments, a Cas protein comprises a
Cas protein of
the Ypest subtype (also known as CASS3). Exemplary Cas proteins of the Ypest
subtype
include, but are not limited to Csyl, Csy2, Csy3, and Csy4. In some
embodiments, a Cas protein
comprises a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary
Cas proteins
of the Nmeni subtype include, but are not limited to Csnl and Csn2. In some
embodiments, a
Cas protein comprises a Cas protein of the Dvulg subtype (also known as
CASS1). Exemplary
Cas proteins of the Dvulg subtype include Csdl, Csd2, and Cas5d. In some
embodiments, a Cas
protein comprises a Cas protein of the Tneap subtype (also known as CASS7).
Exemplary Cas
230
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
proteins of the Tneap subtype include, but are not limited to, Csfl, Cst2,
Cas5t. In some
embodiments, a Cas protein comprises a Cas protein of the Hmari subtype.
Exemplary Cas
proteins of the Hmari subtype include, but are not limited to Cshl, Csh2, and
Cas5h. In some
embodiments, a Cas protein comprises a Cas protein of the Apern subtype (also
known as
CASS5). Exemplary Cas proteins of the Apern subtype include, but are not
limited to Csal,
Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, a Cas protein
comprises a Cas
protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of
the Mtube
subtype include, but are not limited to Csml, Csm2, Csm3, Csm4, and Csm5. In
some
embodiments, a Cas protein comprises a RAMP module Cas protein. Exemplary RAMP
module
Cas proteins include, but are not limited to, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5,
and Cmr6. See,
e.g., Klompe et al., Nature 571,219-225 (2019); Strecker et al., Science
365,48-53 (2019).
Examples of Cas proteins include, but are not limited to: Cas3, Cas8a, Cas5,
Cas8b, Cas8c,
CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and/or GSU0054. In some embodiments, a
Cas protein
comprises Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2,
Csy3, and/or
GSU0054. Examples of Cas proteins include, but are not limited to: Cas9, Csn2,
and/or Cas4. In
some embodiments, a Cas protein comprises Cas9, Csn2, and/or Cas4. In some
embodiments,
Examples of Cas proteins include, but are not limited to: Cas10, Csm2, Cmr5,
Cas10, Csx11,
and/or Csx10. In some embodiments, a Cas protein comprises a Cas10, Csm2,
Cmr5, Cas10,
Csx11, and/or Csx10. In some embodiments, examples of Cas proteins include,
but are not
limited to: Csfl. In some embodiments, a Cos protein comprises Csfl In some
embodiments,
examples of Cas proteins include, but are not limited to: Cas12a, Cas12b,
Cas12c, C2c4, C2c8,
C2c5, C2c10, and C2c9; as well as CasX (Cas12e) and CasY (Cas12d). Also see,
e.g., Koonin et
al., Curr Opin Microbiol. 2017; 37:67-78: "Diversity, classification and
evolution of CRISPR-
Cas systems.- In some embodiments, a Cas protein comprises Cas12a, Cas12b,
Cas12c, Cas12d,
Cas12e, Cas12d, and/or Cas12e. In some embodiments, a Cos protein comprises In
some
embodiments, a Cas protein comprises Cas13, Cas13a, C2c2, Cas13b, Cas13c,
and/or Cas13d.
[00802] In some embodiments, a Cas protein comprises any one of the Cas
proteins described
herein or a functional portion thereof. As used herein, "functional portion"
refers to a portion of
a peptide which retains its ability to complex with at least one ribonucleic
acid (e.g., guide RNA
(gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the
functional
portion comprises a combination of operably linked Cas9 protein functional
domains selected
231
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
from the group consisting of a DNA binding domain, at least one RNA binding
domain, a
helicase domain, and an endonuclease domain. In some embodiments, the
functional portion
comprises a combination of operably linked Cas12a (also known as Cpfl) protein
functional
domains selected from the group consisting of a DNA binding domain, at least
one RNA binding
domain, a helicase domain, and an endonuclease domain. In some embodiments,
the functional
domains form a complex. In some embodiments, a functional portion of the Cas9
protein
comprises a functional portion of a RuvC-like domain. In some embodiments, a
functional
portion of the Cas9 protein comprises a functional portion of the HNH nuclease
domain. In some
embodiments, a functional portion of the Cas12a protein comprises a functional
portion of a
RuvC-like domain.
1008031 In some embodiments, exogenous Cas protein can be introduced into the
cell in
polypeptide form. In certain embodiments, Cas proteins can be conjugated to or
fused to a cell-
penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-
penetrating
polypeptide" and "cell-penetrating peptide" refers to a polypeptide or
peptide, respectively,
which facilitates the uptake of molecule into a cell. The cell-penetrating
polypeptides can contain
a detectable label.
[00804] In certain embodiments, Cas proteins can be conjugated to or fused to
a charged protein
(e.g., that carries a positive, negative or overall neutral electric charge).
Such linkage may be
covalent. In some embodiments, the Cas protein can be fused to a
superpositively charged GFP
to significantly increase the ability of the Cas protein to penetrate a cell
(Cronican et al. ACS
Chem Biol. 2010; 5(8):747-52). In certain embodiments, the Cas protein can be
fused to a
protein transduction domain (PTD) to facilitate its entry into a cell.
Exemplary PTDs include Tat,
oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises
a Cas9
polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9
protein
comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9
protein comprises
a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9
protein comprises a
Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the
Cas9 protein
comprises a Cas9 polypeptide fused to a penetratin domain. In some
embodiments, the Cas9
protein comprises a Cas9 polypeptide fused to a superpositively charged GFP.
In some
embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell-
penetrating
peptide. In some embodiments, the Cas12a protein comprises a Cas12a
polypeptide fused to a
232
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide
fused to a tat
domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide
fused to an
oligoarginine domain. In some embodiments, the Cas12a protein comprises a
Cas12a
polypeptide fused to a penetratin domain. In some embodiments, the Cas12a
protein comprises a
Cas12a polypeptide fused to a superpositively charged GFP.
1008051 In some embodiments, the Cas protein can be introduced into a cell
containing the
target polynucleotide sequence in the form of a nucleic acid encoding the Cas
protein. The
process of introducing the nucleic acids into cells can be achieved by any
suitable technique.
Suitable techniques include calcium phosphate or lipid-mediated transfection,
electroporation,
and transduction or infection using a viral vector. In some embodiments, the
nucleic acid
comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA,
as
described herein. In some embodiments, the nucleic acid comprises mRNA. In
some
embodiments, the nucleic acid comprises a modified mRNA, as described herein
(e.g., a
synthetic, modified mRNA).
1008061 In some embodiments, the Cas protein is complexed with one to two
ribonucleic acids.
In some embodiments, the Cas protein is complexed with two ribonucleic acids.
In some
embodiments, the Cas protein is complexed with one ribonucleic acid. In some
embodiments, the
Cas protein is encoded by a modified nucleic acid, as described herein (e.g.,
a synthetic,
modified mRNA).
1008071 The methods of the present disclosure contemplate the use of any
ribonucleic acid that
is capable of directing a Cas protein to and hybridizing to a target motif of
a target
polynucleotide sequence. In some embodiments, at least one of the ribonucleic
acids comprises
tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises
CRISPR RNA
(crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA
that directs
the Cas protein to and hybridizes to a target motif of the target
polynucleotide sequence in a cell.
In some embodiments, at least one of the ribonucleic acids comprises a guide
RNA that directs
the Cas protein to and hybridizes to a target motif of the target
polynucleotide sequence in a cell.
In some embodiments, both of the one to two ribonucleic acids comprise a guide
RNA that
directs the Cas protein to and hybridizes to a target motif of the target
polynucleotide sequence in
a cell. The ribonucleic acids of the present disclosure can be selected to
hybridize to a variety of
different target motifs, depending on the particular CRISPR/Cas system
employed, and the
233
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
sequence of the target polynucleotide, as will be appreciated by those skilled
in the art. The one
to two ribonucleic acids can also be selected to minimize hybridization with
nucleic acid
sequences other than the target polynucleotide sequence. In some embodiments,
the one to two
ribonucleic acids hybridize to a target motif that contains at least two
mismatches when
compared with all other genomic nucleotide sequences in the cell. In some
embodiments, the one
to two ribonucleic acids hybridize to a target motif that contains at least
one mismatch when
compared with all oilier genomic nucleotide sequences in the cell. In some
embodiments, the one
to two ribonucleic acids are designed to hybridize to a target motif
immediately adjacent to a
deoxyribonucleic acid motif recognized by the Cas protein. In some
embodiments, each of the
one to two ribonucleic acids are designed to hybridize to target motifs
immediately adjacent to
deoxyribonucleic acid motifs recognized by the Cas protein which flank a
mutant allele located
between the target motifs.
[00808] In some embodiments, each of the one to two ribonucleic acids
comprises guide RNAs
that directs the Cas protein to and hybridizes to a target motif of the target
polynucleotide
sequence in a cell.
[00809] In some embodiments, one or two ribonucleic acids (e.g., guide RNAs)
are
complementary to and/or hybridize to sequences on the same strand of a target
polynucleotide
sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs)
are
complementary to and/or hybridize to sequences on the opposite strands of a
target
polynucleotide sequence. In some embodiments, the one or two ribonucleic acids
(e.g., guide
RNAs) are not complementary to and/or do not hybridize to sequences on the
opposite strands of
a target polynucleotide sequence. In some embodiments, the one or two
ribonucleic acids (e.g.,
guide RNAs) are complementary to and/or hybridize to overlapping target motifs
of a target
polynucleotide sequence. In some embodiments, the one or two ribonucleic acids
(e.g., guide
RNAs) are complementary to and/or hybridize to offset target motifs of a
target polynucleotide
sequence.
[00810] In some embodiments, nucleic acids encoding Cas protein and nucleic
acids encoding
the at least one to two ribonucleic acids are introduced into a cell via viral
transduction (e.g.,
lentiviral transduction). In some embodiments, the Cas protein is complexed
with 1-2
ribonucleic acids. In some embodiments, the Cas protein is complexed with two
ribonucleic
acids. In some embodiments, the Cas protein is complexed with one ribonucleic
acid. In some
234
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, the Cos protein is encoded by a modified nucleic acid, as
described herein (e.g., a
synthetic, modified mRNA).
1008111 Exemplary gRNA sequences useful for CRISPR/Cas-based targeting of
genes described
herein are provided in Table 19. The sequences can be found in W02016183041
filed May 9,
2016, the disclosure including the Tables, Appendices, and Sequence Listing is
incorporated
herein by reference in its entirety.
Table 119. Exemplary gRNA sequences useful for targeting genes
Gene Name SEQ ID NO: W02016183041
HLA-A SEQ ID NOs: 2-1418 Table 8, Appendix 1
EILA-B SEQ ID NOs: 1419-3277 Table 9, Appendix 2
HLA-C SEQ ID NOS:3278-5183 Table 10, Appendix 3
RFX-ANK SEQ ID NOs: 95636-102318 Table 11, Appendix 4
NFY-A SEQ ID NOs: 102319-121796 Table 13, Appendix
6
RFX5 SEQ ID NOs: 85645-90115 Table 16, Appendix 9
RFX-AP SEQ ID NOs: 90116-95635 Table 17, Appendix
10
NFY-B SEQ ID NOs. 121797-135112 Table 20, Appendix
13
NFY-C SEQ ID NOs: 135113-176601 Table 22, Appendix
15
IRF1 SEQ ID NOs: 176602-182813 Table 23, Appendix
16
TAP1 SEQ ID NOs: 182814-188371 Table 24, Appendix
17
CIITA SEQ ID NOS:5184-36352 Table 12, Appendix 5
B2M SEQ ID NOS:81240-85644 Table 15, Appendix 8
NLRC5 SEQ ID NOS:36353-81239 Table 14, Appendix 7
CD47 SEQ ID NOS:200784-231885 Table 29, Appendix
22
HLA-E SEQ ID NOS:189859-193183 Table 19, Appendix
12
HLA-F SEQ ID NOS:688808-699754 Table 45, Appendix
38
HLA-G SEQ ID NOS:188372-189858 Table 18, Appendix
11
PD-L1 SEQ ID NOS:193184-200783 Table 21, Appendix
14
Gene Name SEQ ID NO: US20160348073
TRAC SEQ ID NOS: 532-609 and
9102-9797
TRB (also SEQ ID NOS:610-765 and 9798-
TCRB and 10532
TRBC)
1008121 Other exemplary gRNA sequences useful for CRISPR/Cas-based targeting
of genes
described herein are provided in PCT/US22/30394 filed May 20, 2022, the
disclosure of which,
including the Tables, Appendices, and Sequence Listings, is incorporated
herein by reference in
their entireties.
235
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1008131 In some embodiments, the cells of the technology are made using
Transcription
Activator-Like Effector Nucleases (TALEN) methodologies.
1008141 By a "TALE-nuclease" (TALEN) is intended a fusion protein consisting
of a nucleic
acid-binding domain typically derived from a Transcription Activator Like
Effector (TALE) and
one nuclease catalytic domain to cleave a nucleic acid target sequence. The
catalytic domain is
preferably a nuclease domain and more preferably a domain having endonuclease
activity, like
for instance I-TevI, ColE7, NucA and Fok-I. In numerous embodiments, the TALE
domain can
be fused to a meganuclease like for instance I-CreI and I-OnuI or functional
variant thereof. In a
more preferred embodiment, said nuclease is a monomeric TALE-Nuclease. A
monomeric
TALE-Nuclease is a TALE-Nuclease that does not require dimerization for
specific recognition
and cleavage, such as the fusions of engineered TAL repeats with the catalytic
domain of I-TevI
described in W02012138927. Transcription Activator like Effector (TALE) are
proteins from
the bacterial species Xanthomonas comprise a plurality of repeated sequences,
each repeat
comprising di-residues in position 12 and 13 (RVD) that are specific to each
nucleotide base of
the nucleic acid targeted sequence. Binding domains with similar modular base-
per-base nucleic
acid binding properties (MBBBD) can also be derived from new modular proteins
recently
discovered by the applicant in a different bacterial species. The new modular
proteins have the
advantage of displaying more sequence variability than TAL repeats.
Preferably, RVDs
associated with recognition of the different nucleotides are HD for
recognizing C, NG for
recognizing T, NI for recognizing A, NN for recognizing G or A, NS for
recognizing A, C, G or
T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for
recognizing C,
ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for
recognizing G, SN
for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for
recognizing A
or G and SW for recognizing A. In another embodiment, critical amino acids 12
and 13 can be
mutated towards other amino acid residues in order to modulate their
specificity towards
nucleotides A, T, C and G and in particular to enhance this specificity. TALEN
kits are sold
commercially.
1008151 In some embodiments, the cells are manipulated using zinc finger
nuclease (ZFN). A
"zinc finger binding protein" is a protein or polypeptide that binds DNA, RNA
and/or protein,
preferably in a sequence-specific manner, as a result of stabilization of
protein structure through
coordination of a zinc ion. The term zinc finger binding protein is often
abbreviated as zinc
236
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
finger protein or ZFP. The individual DNA binding domains are typically
referred to as
"fingers." A ZFP has least one finger, typically two fingers, three fingers,
or six fingers. Each
finger binds from two to four base pairs of DNA, typically three or four base
pairs of DNA. A
ZFP binds to a nucleic acid sequence called a target site or target segment.
Each finger typically
comprises an approximately 30 amino acid, zinc-chelating, DNA-binding
subdomain. Studies
have demonstrated that a single zinc finger of this class consists of an alpha
helix containing the
two invariant histidine residues co-ordinated with zinc along with the two
cysteine residues of a
single beta turn (see, e.g., Berg & Shi, Science 2711081-1085 (1996)).
[00816] In some embodiments, the cells of the present disclosure are made
using a homing
endonuclease. Such homing endonucleases are well-known to the art (Stoddard
2005). Homing
endonucleases recognize a DNA target sequence and generate a single- or double-
strand break.
Homing endonucleases are highly specific, recognizing DNA target sites ranging
from 12 to 45
base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The
homing endonuclease
according to the technology may for example correspond to a LAGLIDADG
endonuclease, to a
HNH endonuclease, or to a GIY-YIG endonuclease. Preferred homing endonuclease
according
to the present disclosure can be an I-CreI variant.
[00817] In some embodiments, the cells of the technology are made using a
meganuclease.
Meganucleases are by definition sequence-specific endonucleases recognizing
large sequences
(Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-
3774). They can
cleave unique sites in living cells, thereby enhancing gene targeting by 1000-
fold or more in the
vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21,
5034-5040; Rouet et al.,
Mol. Cell. Biol., 1994, 14, 8096-8106; Choulika et al., Mol. Cell. Biol.,
1995, 15, 1968-1973;
Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-5060; Sargent et
al., Mol. Cell. Biol.,
1997, 17, 267-77; Donoho et al., Mol. Cell. Biol, 1998, 18, 4070-4078; Elliott
et al., Mol. Cell.
Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. Biol., 1998, 18,
1444-1448).
1008181 In some embodiments, the cells of the technology are made using RNA
silencing or
RNA interference (RNAi) to knock down (e.g., decrease, eliminate, or inhibit)
the expression of
a polypeptide such as a tolerogenic factor. Useful RNAi methods include those
that utilize
synthetic RNAi molecules, short interfering RNAs (siRNAs), PIWI-interacting
NRAs (piRNAs),
short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other transient knock
down methods
recognized by those skilled in the art. Reagents for RNAi including sequence
specific shRNAs,
237
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
siRNA, miRNAs and the like are commercially available. For instance, CIITA can
be knocked
down in a pluripotent stem cell by introducing a CIITA siRNA or transducing a
CIITA shRNA-
expressing virus into the cell. In some embodiments, RNA interference is
employed to reduce or
inhibit the expression of at least one selected from the group consisting of
CIITA, B2M, NLRC5,
TCR-alpha, and TCR-beta.
1008191 In some embodiments, the cells provided herein are genetically
modified to reduce
expression of one or more immune factors (including target polypeptides) to
create immune-
privileged or hypoimmunogenic cells. In certain embodiments, the cells (e.g.,
stem cells,
induced pluripotent stem cells, differentiated cells, hematopoietic stem
cells, primary T cells and
CAR-T cells) disclosed herein comprise one or more genetic modifications to
reduce expression
of one or more target polynucleotides. Non-limiting examples of such target
polynucleotides and
polypeptides include CIITA, B2M, NLRC5, CTLA-4, PD-1, HLA-A, HLA-BM, HLA-C,
RFX-
ANK, NFY-A, RFX5, RFX-AP, NFY-B, NFY-C, IRF1, and TAP1.
1008201 In some embodiments, the genetic modification occurs using a
CRISPR/Cas system. By
modulating (e.g., reducing or deleting) expression of one or a plurality of
the target
polynucleotides, such cells exhibit decreased immune activation when engrafted
into a recipient
subject. In some embodiments, the cell is considered hypoimmunogenic, e.g., in
a recipient
subject or patient upon administration.
I. Gene editing systems
1008211 In some embodiments, the methods for genetically modifying cells to
knock out, knock
down, or otherwise modify one or more genes comprise using a site-directed
nuclease, including,
for example, zinc finger nucleases (ZFNs), transcription activator-like
effector nucleases
(TALENs), meganucleases, transposases, and clustered regularly interspaced
short palindromic
repeat (CRISPR)/Cas systems, as well as nickase systems, base editing systems,
prime editing
systems, and gene writing systems known in the art.
1. ZFNs
1008221 ZFNs are fusion proteins comprising an array of site-specific DNA
binding domains
adapted from zinc finger-containing transcription factors attached to the
endonuclease domain of
the bacterial FokI restriction enzyme. A ZFN may have one or more (e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9,
10, or more) of the DNA binding domains or zinc finger domains. See, e.g.,
Carroll et al.,
238
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
Genetics Society of America (2011) 188:773-782; Kim et at., Proc. Natl. Acad.
Sci. USA (1996)
93:1156-1160. Each zinc finger domain is a small protein structural motif
stabilized by one or
more zinc ions and usually recognizes a 3- to 4-bp DNA sequence. Tandem
domains can thus
potentially bind to an extended nucleotide sequence that is unique within a
cell's genome.
[00823] Various zinc fingers of known specificity can be combined to produce
multi-finger
polypeptides which recognize about 6, 9, 12, 15, or 18-bp sequences. Various
selection and
modular assembly techniques are available to generate zinc fingers (and
combinations thereof)
recognizing specific sequences, including phage display, yeast one-hybrid
systems, bacterial
one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be
engineered to
bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger
to bind to a
predetermined nucleic acid sequence are known in the art. See, e.g., Sera et
at., Biochemistry
(2002) 41:7074-7081; Liu et al., Bioinfbrmatics (2008) 24:1850-1857.
1008241 ZFNs containing FokI nuclease domains or other dimeric nuclease
domains function as
a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA
sites. The two
individual ZFNs must bind opposite strands of the DNA with their nucleases
properly spaced
apart. See Bitinaite et at., Proc. Natl. Acad. Sci. USA (1998) 95:10570-10575.
To cleave a
specific site in the genome, a pair of ZFNs are designed to recognize two
sequences flanking the
site, one on the forward strand and the other on the reverse strand. Upon
binding of the ZFNs on
either side of the site, the nuclease domains dimerize and cleave the DNA at
the site, generating
a DSB with 5' overhangs. HDR can then be utilized to introduce a specific
mutation, with the
help of a repair template containing the desired mutation flanked by homology
arms. The repair
template is usually an exogenous double-stranded DNA vector introduced to the
cell. See Miller
c/at., Nat. Biotechnol (2011) 29:143-148; Hockemeyer c/at., Nat. Biotechnol.
(2011) 29:731-
734.
TALENs
[00825] TALENs are another example of an artificial nuclease which can be used
to edit a
target gene. TALENs are derived from DNA binding domains termed TALE repeats,
which
usually comprise tandem arrays with 10 to 30 repeats that bind and recognize
extended DNA
sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent
amino acids (termed
the repeat-variable di-residue, or RVD) conferring specificity for one of the
four DNA base
239
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
pairs. Thus, there is a one-to-one correspondence between the repeats and the
base pairs in the
target DNA sequences.
1008261 TALENs are produced artificially by fusing one or more TALE DNA
binding domains
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for
example, a FokI endonuclease
domain. See Zhang, Nature Biotech. (2011) 29:149-153. Several mutations to
FokI have been
made for its use in TALENs; these, for example, improve cleavage specificity
or activity. See
Cermak et al., Nucl. Acids Res. (2011) 39:e82; Miller et al., Nature Biotech.
(2011) 29:143-148;
Hockemeyer et al., Nature Biotech. (2011) 29:731-734; Wood et al., Science
(2011) 333:307;
Doyon et al., Nature Methods (2010) 8:74-79; Szczepek et al, Nature Biotech
(2007) 25:786-
793; Guo et al., J. Mol. Biol. (2010) 200:96. The FokI domain functions as a
dimer, requiring
two constructs with unique DNA binding domains for sites in the target genome
with proper
orientation and spacing. Both the number of amino acid residues between the
TALE DNA
binding domain and the FokI nuclease domain and the number of bases between
the two
individual TALEN binding sites appear to be important parameters for achieving
high levels of
activity. Miller et al., Nature Biotech. (2011) 29:143-148.
1008271 By combining engineered TALE repeats with a nuclease domain, a site-
specific
nuclease can be produced specific to any desired DNA sequence. Similar to
ZFNs, TALENs can
be introduced into a cell to generate DSBs at a desired target site in the
genome, and so can be
used to knock out genes or knock in mutations in similar, HDR-mediated
pathways. See Boch,
Nature Biotech. (2011) 29:135-136; Boch et al. õS'cience (2009) 326:1509-1512;
Moscou etal.,
Science (2009) 326:3501.
111. Meganucleases
1008281 Meganucleases are enzymes in the endonuclease family which are
characterized by
their capacity to recognize and cut large DNA sequences (from 14 to 40 base
pairs).
Meganucleases are grouped into families based on their structural motifs which
affect nuclease
activity and/or DNA recognition. The most widespread and best known
meganucleases are the
proteins in the LAGLIDADG family, which owe their name to a conserved amino
acid sequence.
See Chevalier et al., Nucleic Acids Res. (2001) 29(18): 3757-3774. On the
other hand, the GIY-
YIG family members have a GIY-YIG module, which is 70-100 residues long and
includes four
or five conserved sequence motifs with four invariant residues, two of which
are required for
240
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
activity. See Van Roey et at., Nature Struct. Biol. (2002) 9:806-811. The His-
Cys family
meganucleases are characterized by a highly conserved series of histidines and
cysteines over a
region encompassing several hundred amino acid residues. See Chevalier et at.,
Nucleic Acids
Res. (2001) 29(18):3757-3774. Members of the NT-IN family are defined by
motifs containing
two pairs of conserved histidines surrounded by asparagine residues. See
Chevalier et al.,
Nucleic Acids Res. (2001) 29(18):3757-3774.
1008291 Because the chance of identifying a natural meganuclease for a
particular target DNA
sequence is low due to the high specificity requirement, various methods
including mutagenesis
and high throughput screening methods have been used to create meganuclease
variants that
recognize unique sequences. Strategies for engineering a meganuclease with
altered DNA-
binding specificity, e.g., to bind to a predetermined nucleic acid sequence
are known in the art.
See, e.g., Chevalier et al., Mal. Cell. (2002) 10:895-905; Epinat et at.,
Nucleic Acids Res (2003)
31:2952-2962; Silva et at., J Mol. Biol. (2006) 361:744-754; Seligman et at.,
Nucleic Acids Res
(2002) 30:3870-3879; Sussman et at., J Mol Blot (2004) 342:31-41; Doyon et
at., J Am Chem
Soc (2006) 128:2477-2484; Chen et at., Protein Eng Des Set (2009) 22:249-256;
Arnould et at.,
Mol Biol. (2006) 355:443-458; Smith et al., Nucleic Acids Res. (2006)
363(2):283-294.
1008301 Like ZENs and TALENs, Meganucleases can create DSBs in the genomic
DNA, which
can create a frame-shift mutation if improperly repaired, e.g., via NHEJ,
leading to a decrease in
the expression of a target gene in a cell. Alternatively, foreign DNA can be
introduced into the
cell along with the meganuclease. Depending on the sequences of the foreign
DNA and
chromosomal sequence, this process can be used to modify the target gene. See
Silva et al.,
Current Gene Therapy (2011) 11:11-27.
iv. Transpa.suses
1008311 Transposases are enzymes that bind to the end of a transposon and
catalyze its
movement to another part of the genome by a cut and paste mechanism or a
replicative
transposition mechanism. By linking transposases to other systems such as the
CRISPER/Cas
system, new gene editing tools can be developed to enable site specific
insertions or
manipulations of the genomic DNA. There are two known DNA integration methods
using
transposons which use a catalytically inactive Cas effector protein and Tn7-
like transposons.
241
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
The transposase-dependent DNA integration does not provoke DSBs in the genome,
which may
guarantee safer and more specific DNA integration.
v. CRISPR/Cas systems
1008321 The CRISPR system was originally discovered in prokaryotic organisms
(e.g., bacteria
and archaea) as a system involved in defense against invading phages and
plasmids that provides
a form of acquired immunity. Now it has been adapted and used as a popular
gene editing tool in
research and clinical applications.
1008331 CRISPR/Cas systems generally comprise at least two components: one or
more guide
RNAs (gRNAs) and a Cas protein. The Cas protein is a nuclease that introduces
a DSB into the
target site. CRISPR-Cas systems fall into two major classes: class 1 systems
use a complex of
multiple Cas proteins to degrade nucleic acids; class 2 systems use a single
large Cas protein for
the same purpose. Class 1 is divided into types I, III, and IV; class 2 is
divided into types II, V,
and VI. Different Cas proteins adapted for gene editing applications include,
but are not limited
to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Cas10, Cas12, Cas12a (Cpfl),
Cas12b (C2c1),
Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12f (C2c10), Cas12g, Cas12h,
Cas12i,
Cas12k (C2c5), Cas13, Cas13a (C2c2), Cas13b, Cas13c, Cas13d, C2c4, C2c8, C2c9,
Cmr5,
Csel, Cse2, Csfl, Csm2, Csn2, Csx10, Csx11, Csyl, Csy2, Csy3, and Mad7. The
most widely
used Cas9 is described herein as illustrative. These Cas proteins may be
originated from
different source species. For example, Cas9 can be derived from S. pyogenes or
S. cntrens.
1008341 In the original microbial genome, the type II CRISPR system
incorporates sequences
from invading DNA between CRISPR repeat sequences encoded as arrays within the
host
genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR
RNAs
(crRNAs) each harboring a variable sequence transcribed from the invading DNA,
known as the
"protospacer" sequence, as well as part of the CRISPR repeat. Each ciRNA
hybridizes with a
second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a
complex with the
Cas9 nuclease. The protospacer-encoded portion of the crRNA directs the Cas9
complex to
cleave complementary target DNA sequences, provided that they are adjacent to
short sequences
known as "protospacer adjacent motifs" (PAMs).
242
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1008351 Since its discovery, the CRISPR system has been adapted for inducing
sequence
specific DSBs and targeted genome editing in a wide range of cells and
organisms spanning from
bacteria to eukaryotic cells including human cells. In its use in gene editing
applications,
artificially designed, synthetic gRNAs have replaced the original
crRNA:tracrRNA complex.
For example, the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA,
a
tetraloop, and a tracrRNA. The crRNA usually comprises a complementary region
(also called a
spacer, usually about 20 nucleotides in length) that is user-designed to
recognize a target DNA of
interest. The tracrRNA sequence comprises a scaffold region for Cas nuclease
binding. The
crRNA sequence and the tracrRNA sequence are linked by the tetraloop and each
have a short
repeat sequence for hybridization with each other, thus generating a chimeric
sgRNA. One can
change the genomic target of the Cas nuclease by simply changing the spacer or
complementary
region sequence present in the gRNA. The complementary region will direct the
Cas nuclease to
the target DNA site through standard RNA-DNA complementary base pairing rules.
1008361 In order for the Cas nuclease to function, there must be a PAM
immediately
downstream of the target sequence in the genomic DNA. Recognition of the PAM
by the Cas
protein is thought to destabilize the adjacent genomic sequence, allowing
interrogation of the
sequence by the gRNA and resulting in gRNA-DNA pairing when a matching
sequence is
present. The specific sequence of PAM varies depending on the species of the
Cas gene. For
example, the most commonly used Cas9 nuclease derived from S. pyogenes
recognizes a PAM
sequence of 5'-NGG-3' or, at less efficient rates, 5'-NAG-3', where "N" can be
any nucleotide.
Other Cas nuclease variants with alternative PAMs have also been characterized
and successfully
used for genome editing, which are summarized in Table 20 below.
Table 20. Exemplary Cas nuclease variants and their PAM sequences
CRISPR Nuclease Source Organism PAM Sequence
(5'¨>3')
SpCas9 Streptococcus pyogenes NGG or NAG
SaCas9 Staphylococcus aureus NGRRT or NGRRN
NmeCas9 Neisseria meningitidis NNNNGATT
CjCas9 Campylobacter jejuni NNNNRYAC
StCas9 Streptococcus thermophihts NNAGAAW
TdCas9 Treponema denticola NAAAAC
243
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
LbCas12a (Cpfl) Lachnospiraceae bacterium
TTTV
AsCas12a (Cpfl) Acidaminococcus sp.
TTTV
AacCas12b Alicyclobacillus
acidiphilus TTN
BhCas12b v4 Bacillus hisashii
ATTN, TTTN, or GTTN
R = A or G; Y = C or T; W = A or T; V = A or C or G; N = any base
1008371
In some embodiments, Cas nucleases may comprise one or more mutations to
alter their activity, specificity, recognition, and/or other characteristics.
For example, the Cas
nuclease may have one or more mutations that alter its fidelity to mitigate
off-target effects (e.g.,
eSpCas9, SpCas9-HF1, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity
variants of
SpCas9). For another example the Cas nuclease may have one or more mutations
that alter its
PAM specificity.
vi. Nickases
1008381 Nuclease domains of the Cas, in particular the Cas9, nuclease can be
mutated
independently to generate enzymes referered to as DNA "nickases". Nickases are
capable of
introducing a single-strand cut with the same specificity as a regular
CRISPR/Cas nucleas
system, including for example CRISPR/Cas9. Nickases can be employed to
generate double-
strand breaks which can find use in gene editing systems (Mali et at., Nat
Biotech, 3 1(9):833-838
(2013); Mali et al. Nature Methods, 10:957-963 (2013); Mali et al. õS'cience,
339(6121):823-826
(2013)). In some instances, when two Cas nickases are used, long overhangs are
produced on
each of the cleaved ends instead of blunt ends which allows for additional
control over precise
gene integration and insertion (Mali et at., Nat Biotech, 31(9).833-838
(2013); Mali et al. Nature
Methods, 10:957-963 (2013); Mali c/at., Science, 339(6121).823-826 (2013)). As
both nicking
Cas enzymes must effectively nick their target DNA, paired nickases can have
lower off-target
effects compared to the double-strand-cleaving Cas-based systems (Ran et at.,
Cell, 155(2):479-
480(2013); Mali et al., Nat Biotech, 31(9):833-838 (2013); Mali et al. Nature
Methods, 10:957-
963 (2013); Mali et al, Science, 339(6121):823-826 (2013)).
1008391
244
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
V. Methods of Recombinant Expression of Tolerogenic Factors and/or Chimeric
Antigen
Receptors
1008401 For all of these technologies, well-known recombinant techniques are
used, to generate
recombinant nucleic acids as outlined herein. In certain embodiments, the
recombinant nucleic
acids encoding a tolerogenic factor or a chimeric antigen receptor may be
operably linked to one
or more regulatory nucleotide sequences in an expression construct. Regulatory
nucleotide
sequences will generally be appropriate for the host cell and recipient
subject to be treated.
Numerous types of appropriate expression vectors and suitable regulatory
sequences are known
in the art for a variety of host cells. Typically, the one or more regulatory
nucleotide sequences
may include, but are not limited to, promoter sequences, leader or signal
sequences, ribosomal
binding sites, transcriptional start and termination sequences, translational
start and termination
sequences, and enhancer or activator sequences. Constitutive or inducible
promoters as known
in the art are also contemplated. The promoters may be either naturally
occurring promoters,
hybrid promoters that combine elements of more than one promoter, or synthetic
promoters. An
expression construct may be present in a cell on an episome, such as a
plasmid, or the expression
construct may be inserted in a chromosome such as in a gene locus. In some
embodiment, the
expression vector includes a selectable marker gene to allow the selection of
transformed host
cells. Some embodiments, include an expression vector comprising a nucleotide
sequence
encoding a variant polypeptide operably linked to at least one regulatory
sequence. Regulatory
sequence for use herein include promoters, enhancers, and other expression
control elements. In
some embodiments, an expression vector is designed for the choice of the host
cell to be
transformed, the particular variant polypeptide desired to be expressed, the
vector's copy number,
the ability to control that copy number, and/or the expression of any other
protein encoded by the
vector, such as antibiotic markers.
1008411 Examples of suitable mammalian promoters include, for example,
promoters from the
following genes: elongation factor 1 alpha (EF1a) promoter, CAG promoter,
ubiquitin/S27a
promoter of the hamster (WO 97/1 5664), Simian vacuolating virus 40 (SV40)
early promoter,
adenovirus major late promoter, mouse metallothionein-I promoter, the long
terminal repeat
region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter
(M1VITV),
Moloney murine leukemia virus Long Terminal repeat region, and the early
promoter of human
Cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are
the actin,
245
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
immunoglobulin or heat shock promoter(s). In additional embodiments, promoters
for use in
mammalian host cells can be obtained from the genomes of viruses such as
polyoma virus,
fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus,
avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40
(5V40). In further
embodiments, heterologous mammalian promoters are used. Examples include the
actin
promoter, an immunoglobulin promoter, and heat-shock promoters. The early and
late promoters
of SV40 are conveniently obtained as an SV40 restriction fragment which also
contains the
SV40 viral origin of replication (Fiers eta!, Nature 273: 113-120 (1978)). The
immediate early
promoter of the human cytomegalovirus is conveniently obtained as a HindIII
restriction enzyme
fragment (Greenaway et al, Gene 18: 355-360 (1982)). The foregoing references
are incorporated
by reference in their entirety.
1008421 In some embodiments, the expression vector is a bicistronic or
multicistronic
expression vector. Bicistronic or multicistronic expression vectors may
include (1) multiple
promoters fused to each of the open reading frames; (2) insertion of splicing
signals between
genes; (3) fusion of genes whose expressions are driven by a single promoter;
and (4) insertion
of protcolytic cleavage sites between genes (self-cleavage peptide) or
insertion of internal
ribosomal entry sites (lRESs) between genes.
1008431 The process of introducing the polynucleotides described herein into
cells can be
achieved by any suitable technique. Suitable techniques include calcium
phosphate or lipid-
mediated transfection, electroporation, fusogens, and transduction or
infection using a viral
vector. In some embodiments, the polynucleotides are introduced into a cell
via viral
transduction (e.g., AAV transduction, lentiviral transduction) or otherwise
delivered on a viral
vector (e.g., fusogen-mediated delivery). In some embodiments, the
polynucleotides are
introduced into a cell via a fusogen-mediated delivery or a transposase system
selected from the
group consisting of conditional or inducible transposases, conditional or
inducible PiggyBac
transposons, conditional or inducible Sleeping Beauty (SB11) transposons,
conditional or
inducible Mosl transposons, and conditional or inducible To12 transposons.
1008441 In some embodiments, the cells provided herein are genetically
modified to include one
or more exogenous polynucleotides inserted into one or more genomic loci of
the
hypoimmunogenic cell. In some embodiments, the exogenous polynucleotide
encodes a protein
of interest, e.g., a chimeric antigen receptor. Any suitable method can be
used to insert the
246
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
exogenous polynucleotide into the genomic locus of the hypoimmunogenic cell
including the
gene editing methods described herein (e.g., a CRISPR/Cas system). In some
embodiments, the
exogenous polynucleotide is inserted into at least one allele of the cell
using viral transduction,
for example, with a vector. In some embodiments, the vector is a pseudotyped,
self-inactivating
lentiviral vector that carries the exogenous polynucleotide. In some
embodiments, the vector is a
self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis
VSV-G envelope, and
which catties the exogenous polynucleotide. In some embodiments, the exogenous

polynucleotide is inserted into at least one allele of the cell using viral
transduction. In some
embodiments, the exogenous polynucleotide is inserted into at least one allele
of the cell using a
lentivirus based viral vector.
1008451 Unlike certain methods of introducing the polynucleotides described
herein into cells
which generally involve activating cells, such as activating T cells (e.g.,
CD8+ T cells), suitable
techniques can be utilized to introduce polynucleotides into non-activated T
cells. Suitable
techniques include, but are not limited to, activation of T cells, such as CD8
+ T cells, with one or
more antibodies which bind to CD3, CD8, and/or CD28, or fragments or portions
thereof (e.g.,
scFv and VIM) that may or may not be bound to beads. Surprisingly, fusogen-
mediated
introduction of polynucleotides into T cells is performed in non-activated T
cells (e.g., CD8 + T
cells) that have not been previously contacted with one or more activating
antibodies or
fragments or portions thereof (e.g., CD3, CD8, and/or CD28). In some
embodiments, fusogen-
mediated introduction of polynucleotides into T cells is performed in vivo
(e.g., after the T cells
have been administered to a subject). In other embodiments, fusogen-mediated
introduction of
polynucleotides into T cells is performed in vitro (e.g., before the T cells
are been administered
to a subject).
1008461 Provided herein are non-activated T cells comprising reduced
expression of one or
more Y chromosome genes HLA-A, HLA-B, HLA-C, CIITA, TCR-alpha, and/or TCR-beta

relative to a wild-type T cell, wherein the non-activated T cell further
comprises a first gene
encoding a CD47. In some embodiments, the non-activated T cells comprise
reduced expression
of Protocadherin-11 Y-linked and/or Neuroligin-4 Y-linked, HLA-A, HLA-B, HLA-
C, CIITA,
TCR-alpha, and/or TCR-beta relative to a wild-type T cell, wherein the non-
activated T cell
further comprises a first exogenous polynucleotide encoding a CD47.
247
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00847] In some embodiments, the non-activated T cell has not been treated
with an anti-CD3
antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T
cell costimulatory
molecule. In some embodiments, the non-activated T cell does not express
activation markers. In
some embodiments, the non-activated T cell expresses CD3 and CD28, and wherein
the CD3
and/or CD28 are inactive.
[00848] In some embodiments, the anti-CD3 antibody is OKT3. In some
embodiments, the anti-
CD28 antibody is CD28.2. In some embodiments, the T cell activating cytokine
is selected from
the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and
IL-21. In some
embodiments, the soluble T cell costimulatory molecule is selected from the
group of soluble T
cell costimulatory molecules consisting of an anti-CD28 antibody, an anti-CD80
antibody, an
anti-CD86 antibody, an anti-CD137L antibody, and an anti-ICOS-L antibody.
[00849] In some embodiments, the non-activated T cell is a primary T cell. In
other
embodiments, the non-activated T cell is differentiated from the engineered
and/or
hypoimmunogenic cells of the present disclosure. In some embodiments, the T
cell is a CD8+ T
cell.
[00850] In some embodiments, the first exogenous polynucleotide encodes CD47.
[00851] In some embodiments, the non-activated T cell further comprises a
second exogenous
polynucleotide encoding a chimeric antigen receptor (CAR). In some
embodiments, the CAR is
selected from the group consisting of a CD19-specific CAR, a CD20-specific
CAR, a BCMA-
specific CAR, and a CD22-specific CAR.
[00852] In some embodiments, the first and/or second exogenous polynucleotide
is carried by a
viral vector, including a lentiviral vector. In some embodiments, the first
and/or second
exogenous polynucleotide is carried by a lentiviral vector that comprises a
CD8 binding agent. In
some embodiments, the first and/or second exogenous polynucleotide is
introduced into the cells
using fusogen-mediated delivery or a transposase system selected from the
group consisting of
conditional or inducible transposases, conditional or inducible PiggyBac
transposons, conditional
or inducible Sleeping Beauty (SB11) transposons, conditional or inducible Mosl
transposons,
and conditional or inducible To12 transposons.
1008531 In some embodiments, the first and/or second exogenous polynucleotides
are inserted
into a specific locus of at least one allele of the T cell. In some
embodiments, the specific locus
248
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
is selected from the group consisting of a safe harbor or target locus, a B2M
locus, a CIITA locus,
a TRAC locus, and a TRB locus. In some embodiments, the second exogenous
polynucleotide
encoding the CAR is inserted into the specific locus selected from the group
consisting of a safe
harbor or target locus, a B21v! locus, a CHIA locus, a TRAC locus and a TRB
locus. In some
embodiments, the first exogenous polynucleotide encoding CD47 is inserted into
the specific
locus selected from the group consisting of a safe harbor or target locus, a
B2M locus, a CIITA
locus, a TRAC locus and a TRB locus. In some embodiments, the second exogenous

polynucleotide encoding the CAR and the first exogenous polynucleotide
encoding CD47 are
inserted into different loci. In some embodiments, the second exogenous
polynucleotide
encoding the CAR and the first exogenous polynucleotide encoding CD47are
inserted into the
same locus. In some embodiments, the second exogenous polynucleotide encoding
the CAR and
the first exogenous polynucleotide encoding CD47are inserted into the B2M
locus. In some
embodiments, the second exogenous polynucleotide encoding the CAR and the
first exogenous
polynucleotide encoding CD47are inserted into the CIITA locus. In some
embodiments, the
second exogenous polynucleotide encoding the CAR and the first exogenous
polynucleotideencoding CD47are inserted into the TRAC locus. In some
embodiments, the
second exogenous polynucleotideencoding the CAR and the first exogenous
polynucleotideencoding CD47are inserted into the TRB locus. In some
embodiments, the second
exogenous polynucleotideencoding the CAR and the first exogenous
polynucleotideencoding
CD47are inserted into the safe harbor or target locus. In some embodiments,
the safe harbor or
target locus is selected from the group consisting of a CCR5 gene locus, a
CXCR4 gene locus, a
PPP1R12C gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene
locus, a
Rosa gene locus, an F3 (CD142) gene locus, a MICA gene locus, a MICB gene
locus, a LRP1
(CD91) gene locus, a 1-1MGB1 gene locus, an ABO gene locus, ad RHD gene locus,
a FUT1
locus, and a KDM5D gene locus.
1008541 In some embodiments, the non-activated T cell does not express HLA-A,
HLA-B,
and/or HLA-C antigens. In some embodiments, the non-activated T cell does not
express B2M.
In some embodiments, the non-activated T cell does not express HLA-DP, HLA-DQ,
and/or
HLA-DR antigens. In some embodiments, the non-activated T cell does not
express CIITA. In
some embodiments, the non-activated T cell does not express TCR-alpha. In some
embodiments,
249
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
the non-activated T cell does not express TCR-beta. In some embodiments, the
non-activated T
cell does not express TCR-alpha and TCR-beta.
1008551 In some embodiments, the non-activated T cell is a /vDm yindel/indel,
ALGN4yindel/indel,
B2mindel/indel, cIITAindel/indel, TRAC''' cell comprising the first exogenous
polynucleotide
encoding CD47 and/or the second exogenous polynucleotide encoding CAR inserted
into the
TRAC locus. In some embodiments, the non-activated T cell is a PCDH
lrndevindel ,
NLGN4YindeUnniel ,B2IvPme, CIITAnideumdel , TRACmdellmael cell comprising the
first exogenous
polynucleotide encoding CD47 and the second exogenous polynucleotide encoding
CAR
inserted into the TRAC locus. In some embodiments, the non-activated T cell is
a
PCDI I 1 lYindeinndel NLGN4Yindellindel , B7Mincieliindel CHTAindeVindel
TRACindellindel cell comprising
the first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the TRB locus. In some embodiments, the non-
activated T cell is a
PCDHJ 1 Yi"devind e NLGN4Yi"deuindel, B7111
cHTAindel/indel, TRACi"deufridel cell comprising
the first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into the TRB locus. In some embodiments, the non-
activated T cell is a
PCDH
NLGN4Yindeui"del, B7MErukulel, CIITA1ndeidel, TRACirukuludel cell
comprising
the first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the B2Mlocus. In some embodiments, the non-
activated T cell is a
pcDmiyindellindel, NTG7.T4 yindel/indel, R27Vfindel/inclel CIITAlndel/mael,
TRAcincievinciet cell comprising
the first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into a B2114 locus. In some embodiments, the non-
activated T cell is a
PCDI I 1 1 Yindel/nniel , NLGN4yindel/indel B9M
indel/indel,
TRACindeuindel cell comprising
the first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the CHTA locus. In some embodiments, the non-
activated T cell is a
PCDI I 1 lYincle"del NLGN4Yindel/indel , B71 nde1/incie1 CHTAindel/inciel
TRACindeuindel cell comprising
the first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into a CIITA locus.
1008561 In some embodiments, the non-activated T cell is a PCDH//14ndevindei,
1\TGN4yindel/indel,
B2mind1del, TRBindel/indel cell comprising the first
exogenous polynucleotide
encoding CD47 and/or the second exogenous polynucleotide encoding CAR inserted
into the
TRAC locus. In some embodiments, the non-activated T cell is a
PCDH//Yindeuindel,
250
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
NLGN4Yindevindel, B2Alnclel/indel, CIITAindellindel , TRBindeliinde 1 cell
comprising the first exogenous
polynucleotide encoding CD47 and the second exogenous polynucleotide encoding
CAR
inserted into the J1-?AC locus. In some embodiments, the non-activated T cell
is a
pcDlli yindel/indel NLGN-4y1nde1/inde1 B7A/findebindel, cirmindel/indel,
TRBincigilindel cell comprising the
first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the TRB locus. In some embodiments, the non-
activated T cell is a
PCDHllYnde'el , NLGN4Ymael'el, B 211/pndel/inde , CIITAm e "'del , TRI3md
Umdel cell comprising the
first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into the TRB locus. In some embodiments, the non-
activated T cell is a
PCD1///
NLGN4Yinde'del, B Windel/incle , CHTAindel/indel TRBind61/indel cell
comprising the
first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the B2M locus. In some embodiments, the non-
activated T cell is a
PCD1111171"devind e NLGN4Yi"deuindel, B7Mindel/indel,
, TRBi"deVindel cell comprising the
first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into a B2Mlocus. In some embodiments, the non-activated
T cell is a
PCD1111Yindel/"Iel, NLGN417inde1/1"del, B7Mmieulel,
TRBincw/inclel cell comprising the
first exogenous polynucleotide encoding CD47 and/or the second exogenous
polynucleotide
encoding CAR inserted into the CIITA locus. In some embodiments, the non-
activated T cell is a
pcDH/ yindel/indel N-LGN-4 yindel/indel B?Alindebinclel CIITA"''', TRBin1e1
cell comprising the
first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding CAR inserted into a CIITA locus.
1008571 Provided herein are engineered T cells comprising reduced expression
of HLA-A,
HLA-B, HLA-C, CIITA, TCR-alpha, and/or TCR-beta relative to a wild-type T
cell, wherein the
engineered T cell further comprises a first exogenous polynucleotide encoding
a CD47 carried
by a lentiviral vector that comprises a CD8 binding agent. In some
embodiments, the engineered
T cells comprise reduced expression of Protocadherin-11 Y-linked and/or
Neuroligin-4 Y-linked,
HLA-A, HLA-B, HLA-C, CIITA, TCR-alpha, and/or TCR-beta relative to a wild-type
T cell,
wherein the engineered T cell further comprises a first exogenous
polynucleotide encoding a
CD47 carried by a lentiviral vector that comprises a CD8 binding agent.
1008581 In some embodiments, the engineered T cell is a primary T cell. In
other embodiments,
the engineered T cell is differentiated from the hypoimmunogenic cell of the
present disclosure.
251
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T
cell is a CD4+ T
cell.
1008591 In some embodiments, the engineered T cell does not express activation
markers. In
some embodiments, the engineered T cell expresses CD3 and CD28, and wherein
the CD3
and/or CD28 are inactive.
1008601 In some embodiments, the engineered T cell has not been treated with
an anti-CD3
antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T
cell costimulatory
molecule. In some embodiments, the anti-CD3 antibody is OKT3, wherein the anti-
CD28
antibody is CD28.2, wherein the T cell activating cytokine is selected from
the group of T cell
activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21, and wherein
soluble T cell
costimulatory molecule is selected from the group of soluble T cell
costimulatory molecules
consisting of an anti-CD28 antibody, an anti-CD80 antibody, an anti-CD86
antibody, an anti-
CD137L antibody, and an anti-ICOS-L antibody. In some embodiments, the
engineered T cell
has not been treated with one or more T cell activating cytokines selected
from the group
consisting of IL-2, IL-7, IL-15, and IL-21. In some instances, the cytokine is
IL-2. In some
embodiments, the one or more cytokines is IL-2 and another selected from the
group consisting
of IL-7, IL-15, and IL-21.
1008611 In some embodiments, the non-activated T cell further comprises a
second exogenous
polynucleotide encoding a chimeric antigen receptor (CAR). In some
embodiments, the CAR is
selected from the group consisting of a CD19-specific CAR and a CD22-specific
CAR.
1008621 In some embodiments, the engineered T cell further comprises a second
exogenous
polynucleotide encoding a chimeric antigen receptor (CAR). In some
embodiments, the first
and/or second exogenous polynucleotides are inserted into a specific locus of
at least one allele
of the T cell. In some embodiments, the specific locus is selected from the
group consisting of a
safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a
TRB locus. In some
embodiments, the first exogenous polynucleotide encoding CD47 is inserted into
the specific
locus selected from the group consisting of a safe harbor or target locus, a
B2M locus, a CIITA
locus, a TRAC locus and a TRB locus. In some embodiments, the second exogenous

polynucleotide encoding the CAR is inserted into the specific locus selected
from the group
consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a
TRAC locus and a TRB
locus. In some embodiments, the first exogenous polynucleotide encoding CD47
and the second
252
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
exogenous polynucleotide encoding the CAR are inserted into different loci. In
some
embodiments, the first exogenous polynucleotide encoding CD47 and the second
exogenous
polynucleotide encoding the CAR are inserted into the same locus. In some
embodiments, the
first exogenous polynucleotide encoding CD47 and the second exogenous
polynucleotide
encoding the CAR are inserted into the B2111 locus, the CIITA locus, the TRAC
locus, the TRB
locus, or the safe harbor or target locus. In some embodiments, the safe
harbor or target locus is
selected from the group consisting of a CCR5 gene locus, a CXCR4 gene locus, a
PPP1R12C
gene locus, an albumin gene locus, a SHS231 gene locus, a CLYBL gene locus, a
Rosa gene
locus, an F3 (CD142) gene locus, a MICA gene locus, a MICB gene locus, a LRP1
(CD91) gene
locus, a HMGB1 gene locus, an ABO gene locus, a RHD gene locus, a FUT1 locus,
and a
KDM5D gene locus.
[00863] In some embodiments, the CAR is selected from the group consisting of
a CD19-
specific CAR and a CD22-specific CAR. In some embodiments, the CAR is a CD19-
specific
CAR. In some embodiments, the CAR is a CD22-specific CAR. In some embodiments,
the CAR
comprises an antigen binding domain that binds to any one selected from the
group consisting of
CD19, CD20, CD22, CD38, CD123, CD138, and BCMA.
[00864] In some embodiments, the engineered T cell does not express HLA-A, HLA-
B, and/or
HLA-C antigens, wherein the engineered T cell does not express B2M, wherein
the engineered T
cell does not express HLA-DP, HLA-DQ, and/or HLA-DR antigens, wherein the
engineered T
cell does not express CIITA, and/or wherein the engineered T cell does not
express TCR-alpha
and TCR-beta.
[00865] In some embodiments, the first and/or second exogenous polynucleotides
are inserted
into at least one allele of the T cell using viral transduction. In some
embodiments, the first
and/or second exogenous polynucleotides are inserted into at least one allele
of the T cell using a
lentivirus based viral vector.
1008661 In some embodiments, the engineered T cell is a PCD1-1//Ymdevindei,
NLGN4radevindel,
CIITAI"de"del , TRAC1"deuwdel cell comprising the first exogenous
polynucleotide
encoding CD47 and/or the second exogenous polynucleotide encoding CAR inserted
into the
TRAC locus, into the TRB locus, into the B2Mlocus, or into the CIITA locus. In
some
embodiments, the engineered T cell is a PCDH/Tr"del/'"del, NLGN4Ymalei"del,
B2Mi"delfthdel,
cHTAinclel/mdel, TRBinclel/tridel cell comprising the first exogenous
polynucleotide encoding CD47
253
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
and/or the second exogenous polynucleotide encoding CAR inserted into the TRAC
locus, into
the TRB locus, into the B2111 locus, or into the CIITA locus.
1008671 In some embodiments, the non-activated T cell and/or the engineered T
cell of the
present disclosure are in a subject. In other embodiments, the non-activated T
cell and/or the
engineered T cell of the present disclosure are in vitro.
1008681 In some embodiments, the non-activated T cell and/or the engineered T
cell of the
present disclosure express a CD8 binding agent. In some embodiments, the CD8
binding agent
is an anti-CD8 antibody. In some embodiments, the anti-CD8 antibody is
selected from the group
consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a human
anti-CD8
antibody, a humanized anti-CD8 antibody, a camelid (e.g., llama, alpaca,
camel) anti-CD8
antibody, and a fragment thereof. In some embodiments, the fragment thereof is
an scFy or a
VEIH. In some embodiments, the CD8 binding agent binds to a CD8 alpha chain
and/or a CD8
beta chain.
1008691 In some embodiments, the CD8 binding agent is fused to a transmembrane
domain
incorporated in the viral envelope. In some embodiments, the lentivirus vector
is pseudotyped
with a viral fusion protein. In some embodiments, the viral fusion protein
comprises one or more
modifications to reduce binding to its native receptor.
1008701 In some embodiments, the viral fusion protein is fused to the CD8
binding agent. In
some embodiments, the viral fusion protein comprises Nipah virus F
glycoprotein and Nipah
virus G glycoprotein fused to the CD8 binding agent. In some embodiments, the
lentivirus
vector does not comprise a T cell activating molecule or a T cell
costimulatory molecule. In
some embodiments, the non-activated T cell and/or the engineered T cell of the
present
disclosure are in a subject. In other embodiments, the non-activated T cell
and/or the engineered
T cell of the present disclosure are in vitro.
1008711 In some embodiments, the non-activated rt cell and/or the engineered
rf cell of the
present disclosure express a CD8 binding agent. In some embodiments, the CD8
binding agent
is an anti-CD8 antibody. In some embodiments, the anti-CD8 antibody is
selected from the group
consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a human
anti-CD8
antibody, a humanized anti-CD8 antibody, a camelid (e.g., llama, alpaca,
camel) anti-CD8
antibody, and a fragment thereof. In some embodiments, the fragment thereof is
an scFv or a
254
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
VHH. In some embodiments, the CD8 binding agent binds to a CD8 alpha chain
and/or a CD8
beta chain.
1008721 In some embodiments, the CD8 binding agent is fused to a transmembrane
domain
incorporated in the viral envelope. In some embodiments, the lentivirus vector
is pseudotyped
with a viral fusion protein. In some embodiments, the viral fusion protein
comprises one or more
modifications to reduce binding to its native receptor.
1008731 In some embodiments, the viral fusion protein is fused to the CD8
binding agent. In
some embodiments, the viral fusion protein comprises Nipah virus F
glycoprotein and Nipah
virus G glycoprotein fused to the CD8 binding agent. In some embodiments, the
lentivirus
vector does not comprise a T cell activating molecule or a T cell
costimulatory molecule. In
some embodiments, the lentivirus vector encodes the first exogenous
polynucleotide and/or the
second exogenous polynucleotide.
1008741 In some embodiments, following transfer into a first subject, the non-
activated T cell or
the engineered T cell exhibits one or more responses selected from the group
consisting of (a) a
T cell response, (b) an NK cell response, and (c) a macrophage response, that
are reduced as
compared to a wild-type cell following transfer into a second subject. In some
embodiments, the
first subject and the second subject are different subjects. In some
embodiments, the macrophage
response is engulfment.
1008751 In some embodiments, following transfer into a subject, the non-
activated T cell or the
engineered T cell exhibits one or more selected from the group consisting of
(a) reduced TH1
activation in the subject, (b) reduced NK cell killing in the subject, and (c)
reduced killing by
whole PBMCs in the subject, as compared to a wild-type cell following transfer
into the subject.
1008761 In some embodiments, following transfer into a subject, the non-
activated T cell or the
engineered T cell elicits one or more selected from the group consisting of
(a) reduced donor
specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the
subject, and (c)
reduced complement-dependent cytotoxicity (CDC) in a subject, as compared to a
wild-type cell
following transfer into the subject.
255
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1008771 In some embodiments, the non-activated T cell or the engineered T cell
is transduced
with a lentivirus vector comprising a CD8 binding agent within the subject. In
some
embodiments, the lentivirus vector carries a gene encoding the CAR and/or
CD47.
1008781 In some embodiments, the gene encoding the CAR and/or CD47 is
introduced into the
cells using a gene therapy vector or a transposase system selected from the
group consisting of
transposases, PiggyBac transposons, Sleeping Beauty (SB11) transposons, Mosl
transposons,
and To12 transposons. In some embodiments, the gene therapy vector is a
retrovirus or a
fusosome.
1008791 In some embodiments, following transfer into a first subject, the non-
activated T cell or
the engineered T cell exhibits one or more responses selected from the group
consisting of (a) a
T cell response, (b) an NK cell response, and (c) a macrophage response, that
are reduced as
compared to a wild-type cell following transfer into a second subject. In some
embodiments, the
first subject and the second subject are different subjects. In some
embodiments, the macrophage
response is engulfment.
1008801 In some embodiments, following transfer into a subject, the non-
activated T cell or the
engineered T cell exhibits one or more selected from the group consisting of
(a) reduced TH1
activation in the subject, (b) reduced NK cell killing in the subject, and (c)
reduced killing by
whole PBMCs in the subject, as compared to a wild-type cell following transfer
into the subject.
1008811 In some embodiments, following transfer into a subject, the non-
activated T cell or the
engineered T cell elicits one or more selected from the group consisting of
(a) reduced donor
specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the
subject, and (c)
reduced complement-dependent cytotoxicity (CDC) in a subject, as compared to a
wild-type cell
following transfer into the subject.
1008821 In some embodiments, the non-activated T cell or the engineered T cell
is transduced
with a lentivirus vector comprising a CD8 binding agent within the subject. In
some
embodiments, the lentivirus vector carries a gene encoding the CAR and/or
CD47.
1008831 Provided herein are pharmaceutical compositions comprising a
population of the non-
activated T cells and/or the engineered T cells of the present disclosure and
a pharmaceutically
acceptable additive, carrier, diluent or excipient.
256
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1008841 Provided herein are methods comprising administering to a subject a
composition
comprising a population of the non-activated T cells and/or the engineered T
cells of the present
disclosure , or one or more the pharmaceutical compositions of the present
disclosure .
1008851 In some embodiments, the subject is not administered a T cell
activating treatment
before, after, and/or concurrently with administration of the composition In
some embodiments,
the T cell activating treatment comprises lymphodepletion.
1008861 Provided herein are methods of treating a subject suffeting from
cancel, comprising
administering to a subject a composition comprising a population of the non-
activated T cells
and/or the engineered T cells of the present disclosure, or one or more the
pharmaceutical
compositions of the present disclosure, wherein the subject is not
administered a T cell
activating treatment before, after, and/or concurrently with administration of
the composition. In
some embodiments, the T cell activating treatment comprises lymphodepletion.
1008871 Provided herein are methods for expanding T cells capable of
recognizing and killing
tumor cells in a subject in need thereof within the subject, comprising
administering to a subject
a composition comprising a population of the non-activated T cells and/or the
engineered T cells
of the present disclosure, or one or more the pharmaceutical compositions of
the present
disclosure , wherein the subject is not administered a T cell activating
treatment before, after,
and/or concurrently with administration of the composition. In some
embodiments, the T cell
activating treatment comprises lymphodepl eti on.
1008881 Provided herein are pharmaceutical compositions comprising a
population of the non-
activated T cells and/or the engineered T cells of the present disclosure and
a pharmaceutically
acceptable additive, carrier, diluent or excipient.
1008891 Provided herein are methods comprising administering to a subject a
composition
comprising a population of the non-activated T cells and/or the engineered T
cells of the present
disclosure, or one or more the pharmaceutical compositions of the present
disclosure.
1008901 In some embodiments, the subject is not administered a T cell
activating treatment
before, after, and/or concurrently with administration of the composition. In
some embodiments,
the T cell activating treatment comprises lymphodepletion.
1008911 Provided herein are methods of treating a subject suffering from
autoimmune
diseases/disorders and/or inflammatory diseases/disorders, comprising
administering to a subject
257
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
a composition comprising a population of the non-activated T cells and/or the
engineered T cells
of the present disclosure, or one or more the pharmaceutical compositions of
the present
disclosure, wherein the subject is not administered a T cell activating
treatment before, after,
and/or concurrently with administration of the composition. In some
embodiments, the T cell
activating treatment comprises lymphodepletion.
1008921 Provided herein are methods for expanding T cells capable of
recognizing and killing
tumor cells in a subject in need thereof within the subject, comprising
administering to a subject
a composition comprising a population of the non-activated T cells and/or the
engineered T cells
of the present disclosure, or one or more the pharmaceutical compositions of
the present
disclosure, wherein the subject is not administered a T cell activating
treatment before, after,
and/or concurrently with administration of the composition. In some
embodiments, the T cell
activating treatment comprises lymphodepl eti on.
1008931 Provided herein are dosage regimens for treating a condition, disease
or disorder in a
subject comprising administration of a pharmaceutical composition comprising a
population of
the non-activated T cells and/or the engineered T cells of the present
disclosure, or one or more
the pharmaceutical compositions of the present disclosure, and a
pharmaceutically acceptable
additive, carrier, diluent or excipient, wherein the pharmaceutical
composition is administered in
about 1-3 therapeutically effective doses.
1008941 Provided herein are dosage regimens for treating a condition, disease
or disorder in a
subject comprising administration of a pharmaceutical composition comprising a
population of
the non-activated T cells and/or the engineered T cells of the present
disclosure, or one or more
the pharmaceutical compositions of the present disclosure, and a
pharmaceutically acceptable
additive, carrier, diluent or excipient, wherein the pharmaceutical
composition is administered in
about 1-3 clinically effective doses.
[00895] Once altered, the presence of expression of any of the molecule
described herein can be
assayed using known techniques, such as Western blots, ELISA assays, FACS
assays, other
immunoassays, reverse transcriptase polymerase chain reactions (RT-PCR), and
the like.
258
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
W. Generation of Induced Pluripotent Stem Cells
1008961 The technology provides methods of producing hypoimmunogenic
pluripotent cells. In
some embodiments, the method comprises generating pluripotent stem cells. The
generation of
mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs
for murine cells
or hiPSCs for human cells) is generally known in the art. As will be
appreciated by those in the
art, there are a variety of different methods for the generation of iPCSs. The
original induction
was done from mouse embryonic or adult fibroblasts using the viral
introduction of four
transcription factors, 0ct3/4, Sox2, c-Myc and Klf4; see Takahashi and
Yamanaka Cell 126:663-
676 (2006), hereby incorporated by reference in its entirety and specifically
for the techniques
outlined therein. Since then, a number of methods have been developed; see
Seki et al, World J.
Stem Cells 7(1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri,
editors, Methods
in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer
2013, both of
which are hereby expressly incorporated by reference in their entirety, and in
particular for the
methods for generating hiPSCs (see for example Chapter 3 of the latter
reference).
1008971 Generally, iPSCs are generated by the transient expression of one or
more
reprogramming factors" in the host cell, usually introduced using episomal
vectors. Under these
conditions, small amounts of the cells are induced to become iPSCs (in
general, the efficiency of
this step is low, as no selection markers are used). Once the cells are
"reprogrammed", and
become pluripotent, they lose the episomal vector(s) and produce the factors
using the
endogenous genes.
1008981 As is also appreciated by those of skill in the art, the number of
reprogramming factors
that can be used or are used can vary. Commonly, when fewer reprogramming
factors are used,
the efficiency of the transformation of the cells to a pluripotent state goes
down, as well as the
"pluripotency", e.g., fewer reprogramming factors may result in cells that are
not fully
pluripotent but may only be able to differentiate into fewer cell types.
1008991 In some embodiments, a single reprogramming factor, OCT4, is used. In
other
embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other
embodiments,
three reprogramming factors, OCT4, KLF4 and SOX2, are used. In other
embodiments, four
reprogramming factors, OCT4, KLF4, SOX2 and c-Myc, are used. In other
embodiments, 5, 6 or
7 reprogramming factors can be used selected from SOKMNLT, SOX2, OCT4
(POU5F1),
259
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
KLF4, MYC, NANOG, LIN28, and SV4OL T antigen. In general, these reprogramming
factor
genes are provided on episomal vectors such as are known in the art and
commercially available.
[00900] In general, as is known in the art, iPSCs are made from non-
pluripotent cells such as,
but not limited to, blood cells, fibroblasts, etc., by transiently expressing
the reprogramming
factors as described herein.
X. Assays for Hypoimmunogenicity Phenotypes and Retention of Pluripotency
[00901] Once the engineered and/or hypoimmunogenic cells have been generated,
they may be
assayed for their hypoimmunogenicity and/or retention of pluripotency as is
described in
W02016183041 and W02018132783.
[00902] In some embodiments, hypoimmunogenicity is assayed using a number of
techniques
as exemplified in Figure 13 and Figure 15 of W02018132783. These techniques
include
transplantation into allogeneic hosts and monitoring for hypoimmunogenic
pluripotent cell
growth (e.g., teratomas) that escape the host immune system. In some
instances,
hypoimmunogenic pluripotent cell derivatives are transduced to express
luciferase and can then
followed using bioluminescence imaging. Similarly, the T cell and/or B cell
response of the host
animal to such cells are tested to confirm that the cells do not cause an
immune reaction in the
host animal. T cell responses can be assessed by Elispot, ELISA, FACS, PCR, or
mass
cytometry (CYTOF). B cell responses or antibody responses are assessed using
FACS or
Luminex. Additionally or alternatively, the cells may be assayed for their
ability to avoid innate
immune responses, e.g., NK cell killing, as is generally shown in Figures 14
and 15 of
W02018132783.
[00903] In some embodiments, the immunogenicity of the cells is evaluated
using T cell
immunoassays such as T cell proliferation assays, T cell activation assays,
and T cell killing
assays recognized by those skilled in the art. In some cases, the T cell
proliferation assay
includes pretreating the cells with interferon-gamma and coculturing the cells
with labelled T
cells and assaying the presence of the T cell population (or the proliferating
T cell population)
after a preselected amount of time. In some cases, the T cell activation assay
includes
coculturing T cells with the cells outlined herein and determining the
expression levels of T cell
activation markers in the T cells.
260
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
[00904] In vivo assays can be performed to assess the immunogenicity of the
cells outlined
herein. In some embodiments, the survival and immunogenicity of
hypoimmunogenic cells is
determined using an allogenic humanized immunodeficient mouse model. In some
instances, the
hypoimmunogenic pluripotent stem cells are transplanted into an allogenic
humanized NSG-
SGM3 mouse and assayed for cell rejection, cell survival, and teratoma
formation. In some
instances, grafted hypoimmunogenic pluripotent stem cells or differentiated
cells thereof display
long-term survival in the mouse model.
[00905] Additional techniques for determining immunogenicity including
hypoimmunogenicity
of the cells are described in, for example, Deuse et al., Nature
Biotechnology, 2019, 37, 252-258
and Han et al., Proc Natl Acad Sci USA, 2019, 116(21), 10441-10446, the
disclosures including
the figures, figure legends, and description of methods are incorporated
herein by reference in
their entirety.
[00906] Similarly, the retention of pluripotency is tested in a number of
ways. In some
embodiments, pluripotency is assayed by the expression of certain pluripotency-
specific factors
as generally described herein and shown in Figure 29 of W02018132783.
Additionally or
alternatively, the pluripotent cells are differentiated into one or more cell
types as an indication
of pluripotency.
1009071 As will be appreciated by those in the art, the successful reduction
of the MHC I
function (HLA I when the cells are derived from human cells) in the
pluripotent cells can be
measured using techniques known in the art and as described below; for
example, FACS
techniques using labeled antibodies that bind the HLA complex; for example,
using
commercially available HLA-A, HLA-B, and HLA-C antibodies that bind to the
alpha chain of
the human major histocompatibility HLA Class I antigen molecules.
[00908] In addition, the cells can be tested to confirm that the HLA I complex
is not expressed
on the cell surface. This may be assayed by FACS analysis using antibodies to
one or more
HLA cell surface components as discussed above.
[00909] The successful reduction of the MHC II function (HLA II when the cells
are derived
from human cells) in the pluripotent cells or their derivatives can be
measured using techniques
known in the art such as Western blotting using antibodies to the protein,
FACS techniques, RT-
PCR techniques, etc.
261
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009101 In addition, the cells can be tested to confirm that the HLA II
complex is not expressed
on the cell surface. Again, this assay is done as is known in the art (See
Figure 21 of
W02018132783, for example) and generally is done using either Western Blots or
FACS
analysis based on commercial antibodies that bind to human HLA Class II HLA-
DR, DP and
most DQ antigens.
1009111 In addition to the reduction of one or more HLA I and II (or MHC I and
II) molecules,
the engineered and/or hypoimmunogenic cells of the technology have a reduced
susceptibility to
macrophage phagocytosis and NK cell killing. The resulting hypoimmunogenic
cells -escape"
the immune macrophage and innate pathways due to reduction or lack of the TCR
complex and
the expression of one or more CD47 transgenes.
Y. Exogenous Polynucleotides
1009121 In some embodiments, the engineered and/or hypoimmunogenic cells
provided herein
are genetically modified to include one or more exogenous polynucleotides
inserted into one or
more genomic loci of the hypoimmunogenic cell. In some embodiments, the
exogenous
polynucleotide encodes a protein of interest, e.g., a chimeric antigen
receptor. Any suitable
method can be used to insert the exogenous polynucleotide into the genomic
locus of the
hypoimmunogenic cell including the gene editing methods described herein
(e.g., a CRISPR/Cas
system).
1009131 The exogenous polynucleotide can be inserted into any suitable genomic
loci of the
hypoimmunogenic cell. In some embodiments, the exogenous polynucleotide is
inserted into a
safe harbor or target locus as described herein. Suitable safe harbor and
target loci include, but
are not limited to, a CCR5 gene, a CXCR4 gene, a PPP1R12C (also known as
AAVS1) gene, an
albumin gene, a SHS231 locus, a CLYBL gene, a Rosa gene (e.g., ROSA26), an F3
gene (also
known as CD142), a MICA gene, a MICB gene, a LRP1 gene (also known as CD91), a
HN4GB1
Gene, = an ABO Gene a RHD Gene a FUT1 Gene a PDGFRa Gene an OLIG2 Gene a GFAP
gene, and a KDM5D gene (also known as HY). In some embodiments, the exogenous
polynucleotide is interested into an intron, exon, or coding sequence region
of the safe harbor or
target gene locus. In some embodiments, the exogenous polynucleotide is
inserted into an
endogenous gene wherein the insertion causes silencing or reduced expression
of the endogenous
gene. In some embodiments, the polynucleotide is inserted in a B2M, CIITA,
TRAC, TRB, PD-1
262
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
or CTLA-4 gene locus. Exemplary genomic loci for insertion of an exogenous
polynucleotide
are depicted in Table 21.
Table 21: Exemplary genomic loci for insertion of exogenous polynucleotides
Number species name Ensembl ID Target region Also known as
for cleavage
1 human B2M ENSG00000166710 CDS
2 human CIITA ENSG00000179583 CDS
3 human TRAC ENSG00000277734 CDS
4 human
PPP1R12C ENSG00000125503 Intron 1 and 2 AAVS1
human CLYBL ENSG00000125246 Intron 2
6 human CCR5 ENSG00000160791 Exons 1-3,
introns 1-2,
and CDS
7 human THUMPD3- ENSG00000206573 Intron 1 ROSA26
AS1
8 human Ch- 500 bp SHS231
4:58,976,613 window
9 human F3 ENSG00000117525 CDS CD142
human MICA ENSG00000204520 CDS
11 human MICB ENSG00000204516 CDS
12 human LRP1 ENSG00000123384 CDS
13 human HMGB1 ENSG00000189403 CDS
14 human ABO ENSG00000175164 CDS
human RHD ENSG00000187010 CDS
16 human FUT1 ENSG00000174951 CDS
17 human KDM5D ENSG00000012817 CDS HY
Table 22: Non-limiting examples of Cas9 guide RNAs
SEQ
ID Target
Gene NO: guide sequence PAM site
gRNA cut location
ABO 1 UCUCUCCAUGUGCAGUAGGA AGG Exon 7 chr9:133,257,541
FUT1 2 CUGGAUGUCGGAGGAGUACG CGG Exon 4 chi-19.48,750,822
RH 3 GUCUCCGGAAACUCGAGGUG AGG Exon 2 chr1:25,284,622
F3 (CD142) 4 ACAGUGUAGACUUGAUUGAC GGG Exon 2 chr1:94,540,281
B2M 5 CGUGAGUAAACCUGAAUCUU TGG Exon 2 chr15 : 44,715,434
CIITA 6 GAUAUUGGCAUAAGCCUCCC TGG Exon 3 chr16:10,895,747
TRAC 7 AGAGUCUCUCAGCUGGUACA CGG Exon 1 chr14:22,5547,533
263
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009141 For the Cas9 guides, the spacer sequence for all Cas9 guides is
provided in Table 23,
with description that the 20nt guide sequence corresponds to a unique guide
sequence and can be
any of those described herein, including for example those listed in Table 22.
Table 23: Cas9 guide RNAs
Description SEQ ID NO: Sequence
20 nt guide 8 NNNNNN
sequence*
12 nt 9 GUUUUAGAGCUA
crRNA
repeat
sequence
4 nt 10 GAAA
tetraloop
sequence
64 nt 11 UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU
tracrRNA GAAAAAGUGGCACCGAGUCGGUGCUUU
sequence
Exemplary 12 GUUUUAGAGCUAGAA
full AUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACU
sequence UGAAAAAGUGGCACCGAGUCGGUGCUUU
1009151 In some embodiments, the hypoimmunogenic cell that includes the
exogenous
polynucleotide is derived from a hypoimmunogenic induced pluripotent cell
(HIP), for example,
as described herein. Such hypoimmunogenic cells include, for example, cardiac
cells, neural
cells, cerebral endothelial cells, dopaminergic neurons, glial progenitor
cells, endothelial cells,
thyroid cells, pancreatic islet cells (beta cells), retinal pigmented
epithelium cells, NK cells, and
T cells. In some embodiments, the hypoimmunogenic cell that includes the
exogenous
polynucleotide is a pancreatic beta cell, a T cell (e.g., a primary T cell),
or a glial progenitor cell.
1009161 In some embodiments, the exogenous polynucleotide encodes an exogenous
CD47
polypeptide (e.g., a human CD47 polypeptide) and the exogenous polypeptide is
inserted into a
safe harbor or target gene loci or a safe harbor or target site as disclosed
herein or a genomic
locus that causes silencing or reduced expression of the endogenous gene. In
some embodiments,
the polynucleotide is inserted in a B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene
locus.
1009171 In some embodiments, the hypoimmunogenic cell that includes the
exogenous
polynucleotide is a primary T cell or a T cell derived from a hypoimmunogenic
pluripotent cell
(e.g., a hypoimmunogenic iPSC). In exemplary embodiments, the exogenous
polynucleotide is a
264
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
chimeric antigen receptor (e.g., any of the CARs described herein). In some
embodiments, the
exogenous polynucleotide is operably linked to a promoter for expression of
the exogenous
polynucleotide in the hypoimmunogenic cell.
Z. Pharmaceutically Acceptable Carriers
1009181 In some embodiments, the pharmaceutical composition provided herein
further include
a pharmaceutically acceptable carrier. Acceptable carriers, excipients, or
stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and include buffers
such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic acid and
methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride,
hexamethonium chloride,
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol;
alkyl parabens
such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-
pentanol; and m-cresol);
low molecular weight (less than about 10 residues) polypeptides; proteins,
such as serum
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine, arginine, or
lysine;
monosaccharides, disaccharides, and other carbohydrates including glucose,
mannose, or
dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol,
trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein
complexes); salts
such as sodium chloride; and/or non-ionic surfactants such as polysorbates
(TWEENTm),
poloxamers (PLURONICSTM) or polyethylene glycol (PEG). In some embodiments,
the
pharmaceutical composition includes a pharmaceutically acceptable buffer
(e.g., neutral buffer
saline or phosphate buffered saline).
1009191 In some embodiments, the pharmaceutical composition includes one or
more electrolyte
base solutions selected from the group consisting of lactated CryoStor ,
Ringer's solution,
PlasmaLyte-ATM, Iscove's Modified Dulbecco's Medium, Normosol-RTM, VeenDTM,
Polysal
and Hank's Balanced Salt Solution (containing no phenol red). These base
solutions closely
approximate the composition of extracellular mammalian physiological fluids.
1009201 In some embodiments, the pharmaceutical composition includes one or
more
cryoprotective agents selected from the group consisting of arabinogalactan,
glycerol,
polyvinylpyrrolidone (PVP), dextrose, dextran, trehalose, sucrose, raffinose,
hydroxyethyl starch
(LIES), propylene glycol, human serum albumin (HSA), and dimethylsulfoxide
(DMSO). In
265
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some embodiments, the pharmaceutically acceptable buffer is neutral buffer
saline or phosphate
buffered saline. In some embodiments, pharmaceutical compositions provided
herein include one
or more of CryoStor CSB, Plasma-Lyte-ATM, HSA, DMSO, and trehalose.
1009211 CryoStor is an intracellular-like optimized solution containing
osmotic/oncotic
agents, free radical scavengers, and energy sources to minimize apoptosis,
minimize
ischemia/reperfusion injury and maximize the post-thaw recovery of the
greatest numbers of
viable, functional cells. CryoStor is serum- and protein-free, and non-
immunogenic.
CryoStor is cGMP-manufactured from raw materials of USPgrade or higher.
CryoStor is a
family of solutions pre-formulated with 0%, 2%, 5% or 10% DMSO. CryoStor CSB
is a
DMSO-free version of CryoStor . In some embodiments, the pharmaceutical
composition
includes a base solution of CryoStor CSB at a concentration of about 0-100%,
5-95%, 10-90%,
15-85%, 20-80%, 30-80%, 40-80%, 50-80%, 60-80%, 70-80%, 25-75%, 30-70%, 35-
65%, 40-
60%, or 45-55% w/w. In some embodiments, the pharmaceutical composition
includes a base
solution of CryoStor CSB at a concentration of about 0%, 5%, 10%, 15%, 20%,
25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% w/w.
1009221 PlasmaLyte-ATM is a non-polymeric plasma expander and contains
essential salts and
nutrients similar to those found in culture medium but does not contain
additional constituents
found in tissue culture medium which are not approved for human infusion,
e.g., phenol red, or
are unavailable in U.S.P. grade. PlasmaLyte-ATM contains about 140 mEq/liter
of sodium (Na),
about 5 mEq/liter of potassium (K), about 3 mEq/liter of magnesium (Mg), about
98 mEq/liter of
chloride (Cl), about 27 mEq/liter of acetate, and about 23 mEq/liter of
gluconate. (PlasmaLyte-
ATM is commercially available from Baxter, Hyland Division, Glendale Calif.,
product No.
2B2543). In some embodiments, the pharmaceutical composition includes a base
solution of
PlasmaLyte-ATM at a concentration of about 0-100%, 5-95%, 10-90%, 15-85%, 15-
80%, 15-
75%, 15-70%, 15-65%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-35%, 15-30%,
15-25%,
20-80%, 20-75%, 20-70%, 20-65%, 20-60%, 20-55%, 20-50%, 20-45%, 20-40%, 20-
35%, 20-
30%, 25-75%, 30-70%, 35-65%, 40-60%, or 45-55% w/w. In some embodiments, the
pharmaceutical composition includes a base solution of PlasmaLyte-ATM at a
concentration of
about 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%,
80%, 85%, 90%, 95%, or 100% w/w.
266
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009231 In some embodiments, the pharmaceutical composition includes human
serum albumin
(HSA) at a concentration of about 0-10%, 0.3-9.3%, 0.3-8.3%, 0.3-7.3%, 0.3-
6.3%, 0.3-5.3%,
0.3-4.3%, 0.3-3.3%, 0.3-2.3%, 0.3-1.3%, 0.6-8.3%, 0.9-7.3%, 1.2-6.3%, L5-5.3%,
L8-4.3%, or
2.1-3.3% w/v. In some embodiments, the pharmaceutical composition includes HSA
at a
concentration of about 0%, 0.3%, 0.6%, 0.9%, 1.2%, 1.5%, 1.8%, 2.1%, 2.4%,
2.7%, 3.0%,
3.3%, 3.6%, 3.9%, 4.3%, 4.6%, 4.9%, 5.3%, 5.6%, 5.9%, 6.3%, 6.6%, 6.9%, 7.3%,
7.6%, 7.9%,
8.3%, 8.6%, 8.9%, 9.3%, 9.6%, 9.9%, or 10% w/v.
1009241 In some embodiments, the pharmaceutical composition includes
dimethylsulfoxide
(DMSO) at a concentration of about 0-10%, 0.5-9.5%, 1-9%, 1.5-8.5%, 2-8%, 3-
8%, 4-8%, 5-
8%, 6-8%, 7-8%, 2.5-7.5%, 3-7%, 3.5-6.5%, 4-6%, or 4.5-5.5% v/v. In some
embodiments, the
pharmaceutical composition includes HSA at a concentration of about 0%, 0.25%,
0.5%, 0.75%,
1.0%,1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.5%, 3.75%,
4.0%,
4.25%, 4.5%, 4.75%, 5.0%, 5.25%, 5.5%, 5.75%, 6.0%, 6.25%, 6.5%, 6.75%, 7.0%,
7.25%,
7.5%, 7.75%, 8.0%, 8.25%, 8.5%, 8.75%, 9.0%, 9.25%, 9.5%, 9.75%, or 10.0% v/v.
1009251 In some embodiments, the pharmaceutical composition includes trehalose
at a
concentration of about 0-500 mM, 50-450 mM, 100-400 mM, 150-350 mM, or 200-300
mM. In
some embodiments, the pharmaceutical composition includes trehalose at a
concentration of
about 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100

mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM,
350
mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, or 500 mM.
1009261 Exemplary pharmaceutical composition components are shown in Table 24.

Table 24. Exemplary pharmaceutical composition components.
Formulation Base Solution c[DMS01 Additional
c[trehalosel
c11-1SA1*
A 75% CroStorg 7.5% 0.3%
CSB + 25%
3.75% 0.3%
PlasmaLyte ATM
+ 1.2% HSA 5.3%
0.3% 250 mM
267
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
100% 7.5% 0.3%
PlasmaLyte ATM
5.3%
+ 1 2% HSA
7.5% 5.3% 250 mM
* Additional HSA in addition to PlasmaLyte.
1009271 In some embodiments, the pharmaceutical composition comprises
engineered and/or
hypoimmunogenic cells described herein and a pharmaceutically acceptable
carrier comprising
31.25 % (v/v) Plasma-Lyte A, 31.25 % (v/v) of 5% dextrose/0.45% sodium
chloride, 10%
dextran 40 (LMD)/5% dextrose, 20% (v/v) of 25% human serum albumin (HSA), and
7.5% (v/v)
dimethylsulfoxide (DMSO).
AA. Formulations and Dosage
Regimens
1009281 Any therapeutically effective amount of cells described herein can be
included in the
pharmaceutical composition, depending on the indication being treated. Non-
limiting examples
of the cells include primary T cells, T cells differentiated from
hypoimmunogenic induced
pluripotent stem cells, and other cells differentiated from hypoimmunogenic
induced pluripotent
stem cells described herein. In some embodiments, the pharmaceutical
composition includes at
least about 1 x 102, 5 x 102, 1 x 103, 5 x 103, 1 x 104, 5 x 104, 1 x 105, S x
105, 1 x 106, 5 x 106, 1
x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, 5 x 109, lx 1019, or 5 x 1010
cells. In some
embodiments, the pharmaceutical composition includes up to about 1 x 102, 5 x
102, 1 x 103, 5 x
103, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1
x 108, 5 x 108, 1 x 109, 5
x 109, 1 x 1019, or 5 x 1019 cells. In some embodiments, the pharmaceutical
composition
includes up to about 6.0 x 108 cells. In some embodiments, the pharmaceutical
composition
includes up to about 8.0 x 108 cells. In some embodiments, the pharmaceutical
composition
includes at least about 1 x 102-5 x 102, 5 x 102-1 x 103, 1 x 103-5 x 103, 5 x
103-1 x 104, 1 x 104-
x 104, 5 x 104-1 x 105, 1 x 105-5 x 105, 5 x 105-1 x 106, 1 x 106-5 x 106, 5 x
106-1 x 107, 1 x 107-
5 x 107, 5 x 107-1 x 108, 1 x 108-5 x 108, 5 x 108-1 x 109, 1 x 109-5 x 109, 5
x 109-1 x 1019, or 1 x
1019 - 5 x 1019 cells. In exemplary embodiments, the pharmaceutical
composition includes from
about 1.0 x 106 to about 2.5 x 108 cells. In certain embodiments, the
pharmaceutical composition
includes from about 2.0 x 106 to about 2.0 x 108 cells, such as but not
limited to, primary T cells,
T cells differentiated from hypoimmunogenic induced pluripotent stem cells.
268
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009291 In some embodiments, the pharmaceutical composition has a volume of at
least 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,
120, 130, 140, 150,
160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 ml. In exemplary
embodiments, the
pharmaceutical composition has a volume of up to about 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,
190, 200, 250, 300,
350, 400, or 500 ml. In exemplary embodiments, the pharmaceutical composition
has a volume
of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, or 500 ml. In some
embodiments,
the pharmaceutical composition has a volume of from about 1-50 ml, 50-100 ml,
100-150 ml,
150-200 ml, 200-250 ml, 250-300 ml, 300-350 ml, 350-400 ml, 400-450 ml, or 450-
500 ml. In
some embodiments, the pharmaceutical composition has a volume of from about 1-
50 ml, 50-
100 ml, 100-150 ml, 150-200 ml, 200-250 ml, 250-300 ml, 300-350 ml, 350-400
ml, 400-450
ml, or 450-500 ml. In some embodiments, the pharmaceutical composition has a
volume of from
about 1-10 ml, 10-20 ml, 20-30 ml, 30-40 ml, 40-50 ml, 50-60 ml, 60-70 ml, 70-
80 ml, 70-80 ml,
80-90 ml, or 90-100 ml. In some embodiments, the pharmaceutical composition
has a volume
that ranges from about 5 ml to about 80 ml. In exemplary embodiments, the
pharmaceutical
composition has a volume that ranges from about 10 ml to about 70 ml. In
certain embodiments,
the pharmaceutical composition has a volume that ranges from about 10 ml to
about 50 ml.
1009301 The specific amount/dosage regimen will vary depending on the weight,
gender, age
and health of the individual; the formulation, the biochemical nature,
bioactivity, bioavailability
and the side effects of the cells and the number and identity of the cells in
the complete
therapeutic regimen.
1009311 In some embodiments, a therapeutically effective dose or a clinically
effective dose of
the pharmaceutical composition includes about 1.0 x 105 to about 2.5 x 108
cells at a volume of
about 10 ml to 50 ml and the pharmaceutical composition is administered as a
single
therapeutically effective dose or clinically effective dose. In some cases,
the therapeutically
effective dose or clinically effective dose includes about 1.0 x 105 to about
2.5 x 108 primary T
cells described herein at a volume of about 10 ml to 50 ml. In some cases, the
therapeutically
effective dose or clinically effective dose includes about 1.0 x 105 to about
2.5 x 108 primary T
cells that have been described above at a volume of about 10 ml to 50 ml. In
various cases, the
therapeutically effective dose or clinically effective dose includes about 1.0
x 105 to about 2.5 x
269
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
108 T cells differentiated from hypoimmunogenic induced pluripotent stem cells
described herein
at a volume of about 10 ml to 50 ml. In some embodiments, the therapeutically
effective dose or
clinically effective dose is 1.0 x 1 05, 1.1 x 1 05, 1.2 x 1 05, 1.3 x 1 05,
1.4 x 1 05, 1.5 x 1O, 1.6 x
1 05, 1.7 x 1 05, 1.8 x 1 05, 1.9 x 1 05, 2.0 x 1 05, 2.1 x 1 05, 2.2 x 1 05,
2.3 x 105, 2.4 x 105, 2.5 x 1 05,
1.0 x 106, 1.1 x 106, 1.2 x 106, 1.3 x 106, 1.4x 106, 1.5 x 106, 1.6x 106, 1.7
x 106, 1.8 x 106, 1.9x
106, 2.0 x 106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 1.0 x 1
07, 1.1 x 1 07, 1.2 x 1 07,
1.3 x 1 07, 1.4 x 1 07, 1.5 x 1 07, 1.6 x 1 07, 1.7 x 1 07, 1.8 x 107, 1.9 x 1
07, 2.0 x 1 07, 2.1 x 1 07, 2.2 x
107, 2.3 x 107, 2.4 x 107, 2.5 x 107, 1.0 x 108, 1.1 x 108, 1.2 x 108, 1.3 x
108, 1.4 x i0, 1.5 x 108,
1.6 x 1 08, 1.7 x 1 08, 1.8 x 1 08, 1.9 x 108, 2.0 x 108, 2.1 x 108, 2.2 x 1
08, 2.3 x 108, 2.4 x 10, or
2.5 x 108 T cells differentiated from hypoimmunogenic induced pluripotent stem
cells described
herein at a volume of about 1 0 ml to 50 ml. In other cases, the
therapeutically effective dose or
clinically effective dose is at a range that is lower than about 1.0 x i05 to
about 2.5 x 108 T cells,
including primary T cells or T cells differentiated from hypoimmunogenic
induced pluripotent
stem cells. In yet other cases, the therapeutically effective dose or
clinically effective dose is at a
range that is higher than about 1.0 x 1 05 to about 2.5 x 108 T cells,
including primary T cells and
T cells differentiated from hypoimmunogenic induced pluripotent stem cells.
1009321 In some embodiments, the pharmaceutical composition is administered as
a single
therapeutically effective dose or clinically effective dose of from about 1.0
x 105 to about 1.0 x
1 07 cells (such as primary T cells and T cells differentiated from
hypoimmunogenic induced
pluripotent stem cells) per kg body weight for subjects 50 kg or less. In some
embodiments, the
pharmaceutical composition is administered as a single therapeutically
effective dose or
clinically effective dose of from about 0.5 x 1 05 to about 1.0 x 1 07, about
1.0 x 1 05 to about 1.0 x
1 07, about 1.0 x 1 05 to about 1.0 x 1 07, about 5.0 x 1 05 to about 1 x 10,
about 1.0 x 1 06 to about
1 x i07, about 5.0 x 106 to about 1.0 x i07, about 1.0 x i05 to about 5.0 x
106, about 1.0 x i05 to
about 1.0 x 106, about 1.0 x i05 to about 5.0 x i05, about 1.0 x 1 05 to about
5.0 x 106, about 2.0 x
1 05 to about 5.0 x 106, about 3.0 x 1 05 to about 5.0 x 106, about 4.0 x 1 05
to about 5.0 x 106,
about 5.0 x 1 05 to about 5.0 x 106, about 6.0 x 105 to about 5.0 x 106, about
7.0 x 105 to about 5.0
x 106, about 8.0 x 1 05 to about 5.0 x 106, or about 9.0 x 1 05 to about 5.0 x
106 cells per kg body
weight for subjects 50 kg or less. In some embodiments, the therapeutically
effective dose or
clinically effective dose is 0.5 x 105, 0.6 x 105, 0.7 x 105, 0.8 x 105, 0.9 x
105, 1.0 x 105, 1.1 x
105, 1.2 x 105, 1.3 x 105, 1.4 x 105, 1.5 x 10, 1.6 x 105, 1.7 x 105, 1.8 x
105, 1.9 x 105, 2.0 x 105,
270
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
2.1 x 105, 2.2 x 105, 2.3 x 105, 2.4 x 105, 2.5 x 105, 2.6 x 105, 2.7 x 105,
2.8 x 105, 2.9 x 105, 3.0 x
105, 3.1 x 105, 3.2 x 105, 3.3 x 105, 3.4 x 10, 3.5 x 105, 3.6 x 105, 3.7 x
105, 3.8 x 105, 3.9 x 105,
4.0 x 105, 4.1 x 105, 4.2 x 105, 4.3 x 105, 4.4 x 105, 4.5 x 105, 4.6 x 105,
4.7 x 105, 4.8 x 105, 4.9 x
105, 5.0 x 105, 0.5x 106, 0.6x 106, 0,7x 106, 0.8x 106, 0.9x 106, 1.0 x 106,
1.1 x 106, 1.2x 106,
1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106,
2.0 x 106, 2.1 x 106, 2.2 x
106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x
106, 3.0 x 106, 3.1 x 106,
3.2 x 106, 3.3 x 106 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106 3.9
x 106, 4.0 x 106, 4.1 x
106, 4.2 x 106, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x
106, 4.9 x 106, 5.0 x 106,
5.1 x 106, 5.2 x 106 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x 106, 5.7 x 106 5.8
x 106, 5.9 x 106, 6.0 x
106, 6.1 x 106, 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x 106, 6.6 x 106, 6.7 x
106, 6.8 x 106, 6.9 x 106,
7.0 x 106, 7.1 x 106 7.2 x 106, 7.3 x 106, 7.4 x 106, 7.5 x 106, 7.6 x 106 7.7
x 106, 7.8 x 106, 7.9 x
106, 8.0 x 106, 8.1 x 106, 8.2 x 106, 8.3 x 106, 8.4 x 106, 8.5 x 106, 8.6 x
106, 8.7 x 106, 8.8 x 106,
8.9 x 106, 9.0 x 106 9.1 x 106, 9.2 x 106, 9.3 x 106, 9.4 x 106, 9.5 x 106,
9.6 x 106, 9.7 x 106, 9.8 x
106, 9.9 x 106, 0.5 x 107, 0.6 x 107, 0.7 x 107, 0.8 x 107, 0.9 x 107, or 1.0
x 107 cells per kg body
weight for subjects 50 kg or less. In some embodiments, the therapeutically
effective dose or
clinically effective dose is from about 0.2 x 106 to about 5.0 x 106 cells per
kg body weight for
subjects 50 kg or less. In certain embodiments, the therapeutically effective
dose or clinically
effective dose is at a range that is lower than from about 0.2 x 106 to about
5.0 x 106 cells per kg
body weight for subjects 50 kg or less, or clinically effective dose In
exemplary embodiments,
the single therapeutically effective dose or clinically effective dose is at a
volume of about 10 ml
to 50 ml. In some embodiments, the therapeutically effective dose or
clinically effective dose is
administered intravenously.
1009331 In exemplary embodiments, the cells are administered in a single
therapeutically
effective dose of from about 1.0 x 106 to about 5.0 x 108 cells (such as
primary T cells and T cells
differentiated from hypoimmunogenic induced pluripotent stem cells) for
subjects above 50 kg.
In some embodiments, the pharmaceutical composition is administered as a
single
therapeutically effective dose or clinically effective dose of from about 0.5
x 106 to about 1.0 x
109, about 1.0 x 106 to about 1.0 x 109, about 1.0 x 106 to about 1.0 x 109,
about 5.0 x 106 to
about 1.0 x 109, about 1.0 x 107 to about 1.0 x 109, about 5.0 x 107 to about
1.0 x 109, about 1.0 x
106 to about 5.0 x 107, about 1.0 x 106 to about 1.0 x 107, about 1.0 x 106 to
about 5.0 x 107,
about 1.0 x 107 to about 5.0 x 108, about 2.0 x 107 to about 5.0 x 108, about
3.0 x 107 to about 5.0
271
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
x 108, about 4.0 x 107 to about 5.0 x 108, about 5.0 x 107 to about 5.0 x 108,
about 6.0 x 107 to
about 5.0 x 108, about 7.0 x 107 to about 5.0 x 108, about 8.0 x 107 to about
5.0 x 108, or about
9.0 x 107 to about 5.0 x 108 cells per kg body weight for subjects 50 kg or
less. In some
embodiments, the therapeutically effective dose or clinically effective dose
is 1.0 x 106, 1.1 x
106, 1.2 x 106, 1.3 x 106, 1.4 x 106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x
106, 1.9 x 106, 2.0 x 106,
2.1 x 106, 2.2 x 106 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106,
2.8 x 106, 2.9 x 106, 3.0 x
106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4x 106, 3.5 x 106, 3.6 x 106, 3.7 x
106, 3.8 x 106, 3.9 x 106,
4.0 x 106, 4.1 x 106 4.2 x 106, 4.3 x 106, 4.4 x 106, 4.5 x 106, 4.6 x 106,
4.7 x 106, 4.8 x 106, 4.9 x
106, 5.0 x 106, 5.1 x 106, 5.2 x 106, 5.3 x 106, 5.4 x 106, 5.5 x 106, 5.6 x
106, 5.7 x 106, 5.8 x 106,
5.9 x 106, 6.0 x 106 6.1 x 106, 6.2 x 106, 6.3 x 106, 6.4 x 106, 6.5 x 106,
6.6 x 106, 6.7 x 106, 6.8 x
106, 6.9 x 106, 7.0 x 106, 7.1 x 106, 7.2 x 106, 7.3 x 106, 7.4 x 106, 7.5 x
106, 7.6 x 106, 7.7 x 106,
7.8 x 106, 7.9 x 106 8.0 x 106, 8.1 x 106, 8.2 x 106, 8.3 x 106, 8.4 x 106,
8.5 x 106, 8.6 x 106, 8.7 x
106, 8.8 x 106, 8.9 x 106, 9.0 x 106, 9.1 x 106, 9.2 x 106, 9.3 x 106, 9.4 x
106, 9.5 x 106, 9.6 x 106,
9.7x 106, 9.8x 106 9.9x 106, 1.0 x 107, 1.1 x 107, 1.2x 107, 1.3 x 107, 1.4x
107, 1.5 x 107, 1.6x
107, 1.7 x 107, 1.8 x 107, 1.9 x 107, 2.0 x 107, 2.1 x 107, 2.2 x 107, 2.3 x
107, 2.4 x 107, 2.5 x 107,
2.6 x 107, 2.7 x 107 2.8 x 107, 2.9 x 107, 3.0 x 107, 3.1 x 107, 3.2 x 107,
3.3 x 107, 3.4 x 107, 3.5 x
107, 3.6 x 107, 3.7 x 107, 3.8 x 107, 3.9 x 107, 4.0 x 107, 4.1 x 107, 4.2 x
107, 4.3 x 107, 4.4 x 107,
4.5 x 107, 4.6 x 107, 4.7 x 107, 4.8 x 107, 4.9 x 107, 5.0 x 107, 5.1 x 107,
5.2 x 107, 5.3 x 10-7, 5.4 x
107, 5.5 x 107, 5.6x 107, 5.7x 107, 5,8x 107, 5.9x 107, 6.0 x 107, 6.1 x 107,
6.2x 107, 6.3 x 107,
6.4 x 107, 6.5 x 107, 6.6 x 107, 6.7 x 107, 6.8 x 107, 6.9 x 107, 7.0 x 107,
7.1 x 107, 7.2 x 107, 7.3 x
107, 7.4 x 107, 7.5 x 107, 7.6 x 107, 7.7 x 107, 7.8 x 107, 7.9 x 107, 8.0 x
107, 8.1 x 107, 8.2 x 107,
8.3 x 107, 8.4 x 107, 8.5 x 107, 8.6 x 107, 8.7 x 107, 8.8 x 107, 8.9 x 107,
9.0 x 10, 9.1 x 107, 9.2 x
107, 9.3 x 107, 9.4 x 107, 9.5 x 107, 9.6 x 107, 9.7 x 107, 9.8 x 107, 9.9 x
107, 1.0 x 108, 1.1 x 108,
1.2 x 108, 1.3 x 108, 1.4 x 108, 1.5 x 108, 1.6 x 108, 1.7 x 108, 1.8 x 108,
1.9 x 108, 2.0 x 108, 2.1 x
108, 2.2 x 108, 2.3 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.7 x 108, 2.8 x
108, 2.9 x 108, 3.0 x 108,
3.1 x 108, 3.2 x 108, 3.3 x 108, 3.4 x 108, 3.5 x 108, 3.6 x 108, 3.7 x 108,
3.8 x 108, 3.9 x 108, 4.0 x
108, 4.1 x 108, 4.2 x 108, 4.3 x 108, 4.4 x 108, 4.5 x 108, 4.6 x 108, 4.7 x
108, 4.8 x 108, 4.9 x 108,
or 5.0 x 108 cells per kg body weight for subjects 50 kg or less. In certain
embodiments, the cells
are administered in a single therapeutically effective dose or clinically
effective dose of about 1.0
x 107 to about 2.5 x 108 cells for subjects above 50 kg. In some embodiments,
the cells are
administered in a single therapeutically effective dose or clinically
effective dose of a range that
272
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
is less than about 1.0 x 107 to about 2.5 x 108 cells for subjects above 50
kg. In some
embodiments, the cells are administered in a single therapeutically effective
dose or clinically
effective dose of a range that is higher than about 1.0 x 107 to about 2.5 x
108 cells for subjects
above 50 kg. In some embodiments, the dose is administered intravenously. In
exemplary
embodiments, the single therapeutically effective dose or clinically effective
dose is at a volume
of about 10 ml to 50 ml. In some embodiments, the therapeutically effective
dose or clinically
effective dose is administered intravenously.
[00934] In exemplary embodiments, the therapeutically effective dose or
clinically effective
dose is administered intravenously at a rate of about 1 to 50 ml per minute, 1
to 40 ml per
minute, 1 to 30 ml per minute, 1 to 20 ml per minute, 10 to 20 ml per minute,
10 to 30 ml per
minute, 10 to 40 ml per minute, 10 to 50 ml per minute, 20 to 50 ml per
minute, 30 to 50 ml per
minute, 40 to 50 ml per minute. In numerous embodiments, the pharmaceutical
composition is
stored in one or more infusion bags for intravenous administration. In some
embodiments, the
dose is administered completely at no more than 10 minutes, 15 minutes, 20
minutes, 25
minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 60 minutes,
70 minutes, 80 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 240
minutes, or 300
minutes.
[00935] In some embodiments, a single therapeutically effective dose or
clinically effective
dose of the pharmaceutical composition is present in a single infusion bag. In
other
embodiments, a single therapeutically effective dose or clinically effective
dose of the
pharmaceutical composition is divided into 2, 3, 4 or 5 separate infusion
bags.
[00936] In some embodiments, the cells described herein are administered in a
plurality of
doses such as 2, 3, 4, 5, 6 or more doses, wherein the plurality of doses
together constitute a
therapeutically effective dose or clinically effective dose regimen. In some
embodiments, each
dose of the plurality of doses is administered to the subject ranging from 1
to 24 hours apart. In
some instances, a subsequent dose is administered from about 1 hour to about
24 hours (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or about 24
hours) after an initial or preceding dose. In some embodiments, each dose of
the plurality of
doses is administered to the subject ranging from about 1 day to 28 days
apart. In some
instances, a subsequent dose is administered from about 1 day to about 28 days
(e.g., about 1, 2,
273
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, or about 28
days) after an initial or preceding dose. In certain embodiments, each dose of
the plurality of
doses is administered to the subject ranging from 1 week to about 6 weeks
apart. In certain
instances, a subsequent dose is administered from about 1 week to about 6
weeks (e.g., about 1,
2, 3, 4, 5, or 6 weeks) after an initial or preceding dose. In several
embodiments, each dose of
the plurality of doses is administered to the subject ranging from about 1
month to about 12
months apart. In several instances, a subsequent dose is administered from
about 1 month to
about 12 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months)
after an initial or
preceding dose.
1009371 In some embodiments, a subject is administered a first dosage regimen
at a first
timepoint, and then subsequently administered a second dosage regimen at a
second timepoint.
In some embodiments, the first dosage regimen is the same as the second dosage
regimen. In
other embodiments, the first dosage regimen is different than the second
dosage regimen. In
some instances, the number of cells in the first dosage regimen and the second
dosage regimen
are the same. In some instances, the number of cells in the first dosage
regimen and the second
dosage regimen are different. In some cases, the number of doses of the first
dosage regimen
and the second dosage regimen are the same. In some cases, the number of doses
of the first
dosage regimen and the second dosage regimen are different.
1009381 In some embodiments, the first dosage regimen includes hypoimmune
(HIP) T cells or
primary T cells expressing a first CAR and the second dosage regimen includes
hypoimmune
(HIP) T cells or primary T cells expressing a second CAR such that the first
CAR and the second
CAR are different. For instance, the first CAR and second CAR bind different
target antigens. In
some cases, the first CAR includes an scFy that binds an antigen and the
second CAR includes
an scFv that binds a different antigen. In some embodiments, the first dosage
regimen includes
hypoimmune (HIP) T cell or primary T cells expressing a first CAR and the
second dosage
regimen includes hypoimmune (HIP) T cell or primary T cells expressing a
second CAR such
that the first CAR and the second CAR are the same. The first dosage regimen
can be
administered to the subject at least 1 month, 2 months, 3 months, 4 months, 5
months, 6 months,
7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1-3 months, 1-6
months, 4-6
months, 3-9 months, 3-12 months, or more months apart from the second dosage
regimen. In
274
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
some embodiments, a subject is administered a plurality of dosage regimens
during the course of
a disease (e.g., autoimmune diseases) and at least two of the dosage regimens
comprise the same
type of hypoimmune (HIP) T cells or primary T cells described herein. In other
embodiments, at
least two of the plurality of dosage regimens comprise different types of
hypoimmune (HIP) T
cells or primary T cells described herein.
1009391 In some embodiments, the CD19 specific (CD19) CAR-T cells described
herein are
administered to a subject at a dose of about 50 x 106 to about 110 x 106
(e.g., 50 x 106, 51 x 106,
52 x 106, 53 x 106, 54 x 106, 55 x 106, 56 x 106, 57 x 106, 58 x 106, 59 x
106, 60 x 106, 61 x 106,
62 x 106, 63 x 106, 64 x 106, 65 x 106, 66 x 106, 67 x 106, 68 x 106, 69 x
106, 70 x 106, 71 x 106,
72 x 106, 73 x 106, 74 x 106, 75 x 106, 76 x 106, 77 x 106, 78 x 106, 79 x
106, 80 x 106, 81 x 106,
82 x 106, 83 x 106, 84 x 106, 85 x 106, 86 x 106, 87 x 106, 88 x 106, 89 x
106, 90 x 106, 91 x 106,
92 x 106, 93 x 106, 94 x 106, 95 x 106, 96 x 106, 97 x 106, 98 x 106, 99 x
106, 100 x 106, 101 x
106, 102 x 106, 103 x 106, 104 x 106, 105 x 106, 106 x 106, 107 x 106, 108 x
106, 109 x 106, or
110 x 106) viable CD19 specific CAR-T cells. In some embodiments, the dose is
a
therapeutically effective amount of viable CD19 specific CAR-T cells. In other
embodiments,
the dose is a clinically effective amount of viable CD19 specific CAR-T cells.
In some
embodiments, the viable CD19 specific CAR-T cells include CD19 specific CAR
expressing
CD4+ T cells and CD19 specific CAR expressing CD8+ T cells at a ratio of about
1:1. In some
embodiments, the CD19 specific CAR of the cells is lisocabtagene maraleucel
(BREYANZI ), a
structural equivalent thereof, or a functional equivalent thereof.
1009401 In some embodiments, a subject is administered about 50 x 106 to about
110 x 106 (e.g.,
50 x 106, 51 x 106, 52 x 106, 53 x 106, 54 x 106, 55 x 106, 56 x 106, 57 x
106, 58 x 106, 59 x 106,
60 x 106, 61 x 106, 62 x 106, 63 x 106, 64 x 106, 65 x 106, 66 x 106, 67 x
106, 68 x 106, 69 x 106,
70 x 106, 71 x 106, 72 x 106, 73 x 106, 74 x 106, 75 x 106, 76 x 106, 77 x
106, 78 x 106, 79 x 106,
80 x 106, 81 x 106, 82 x 106, 83 x 106, 84 x 106, 85 x 106, 86 x 106, 87 x
106, 88 x 106, 89 x 106,
90 x 106, 91 x 106, 92 x 106, 93 x 106, 94 x 106, 95 x 106, 96 x 106, 97 x
106, 98 x 106, 99 x 106,
100 x 106, 101 X 106, 102 x 106, 103 x 106, 104 x 106, 105 X 106, 106 x 106,
107 x 106, 108 x 106,
109 x 106, or 110 x 106) viable CD19 specific CAR-T cells described herein. In
some
embodiments, the dose is a therapeutically effective amount of viable CD19
specific CAR-T
cells. In other embodiments, the dose is a clinically effective amount of
viable CD19 specific
275
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
CAR-T cells. In some instances, 50% of the viable CD19 specific CAR-T cells
are CD19
specific CAR expressing CD4+ T cells and 50% of the viable CD19 specific CAR-T
cells are
CD19 specific CAR expressing CD8+ T cells. In some embodiments, the CD19
specific CAR of
the cells is lisocabtagene maraleucel (BREYANZIc)), a structural equivalent
thereof, or a
functional equivalent thereof.
1009411 In some embodiments, the CD19 specific CAR-T cells described herein
are
administered to a subject at a dose of about 2 x 106 per kg of body weight. In
some
embodiments, a maximum dose administered is about 2 x 108 viable CD19 specific
CAR-T cells.
In some embodiments, the dose is a therapeutically effective amount of viable
CD19 specific
CAR-T cells. In other embodiments, the dose is a clinically effective amount
of viable CD19
specific CAR-T cells. In some embodiments, the CD19 specific CAR of the cells
is the same
CD19 specific CAR as axicabtagene ciloleucel (YESCARTA ), a structural
equivalent thereof,
or a functional equivalent thereof.
1009421 In some embodiments, the CD19 specific CAR-T cells described herein
are
administered to a subject at a dose of about 2 x 106 per kg of body weight. In
some
embodiments, a maximum dose of about 2 x 108 viable CD19 specific CAR-T cells
is
administered to a patient of about 100 kg of body weight and above. In some
embodiments, the
dose is a therapeutically effective amount of viable CD19 specific CAR-T
cells. In other
embodiments, the dose is a clinically effective amount of viable CD19 specific
CAR-T cells. In
some embodiments, the CD19 specific CAR of the cells is the same CD19 specific
CAR as
brexucabtagene autoleucel (TECARTUS*), a structural equivalent thereof, or a
functional
equivalent thereof.
1009431 In some embodiments, the CD19 specific CAR-T cells described herein
are
administered to a subject at a dose of up to about 2 x 108 viable CD19
specific CAR-T cells. In
some embodiments, a subject is administered from about 0.2 x 106 to about 5.0
x 106 (e.g., about
0.2 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.8 X 106, 0.9 x 106, 1.0 x 106,
1.2 x 106, 1.4 x 106, 1.5 x
106, 1.6 x 106, 1.8 x 106, 1.9 x 106, 2.0 x 106, 2.2 x 106, 2.4 x 106, 2.5 x
106, 2.6 x 106, 2.8 x 106,
2.9 x 106, 3.0 x 106, 3.2 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.8 x 106,
3.9 x 106, 4.0 x 106, 4.2 x
106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.8 x 106, 4.9 x 106, or 5.0 x 106)
viable CD19 specific CAR-
T cells per kg of body weight for a subject with a body weight of about 50 kg
or less. In some
276
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, a subject is administered from about 0.1 x 108 to about 2.5 x 108
(e.g., about 0.1 x
106, 0.2 x 106, 0.4 x 106, 0.5 x 106, 0.6 x 106, 0.8 x 106, 0.9 x 106, 1.0 x
106, 1.2 x 106, 1.4 x 106,
1.5 x 106, 1.6 x 106, 1.8 x 106, 1.9 x 106, 2.0 x 106, 2.2 x 106, 2.4 x 106,
or 2.5 x 106) viable CD19
specific CAR-T cells fora subject with a body weight of greater than about 50
kg. In some
embodiments, a subject is administered from about 0.6 x 108 to about 6.0 x 108
(e.g., about 0.6 x
108, 0.8 x 108, 0.9 x 108, 1.0 x 108, 1.2 x 108, 1.4 x 108, 1.5 x 108, 1.6 x
108, 1.8 x 108, 1.9 x 108,
2.0 x 108, 2.2 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.8 x 108, 2.9 x 108,
3.0 x 108, 3.2 x 108, 3.4 x
108, 3.5 x 108, 3.6 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.2 x 108, 4.4 x
108, 4.5 x 108, 4.6 x 108,
4.8 x 108, 4.9 x 108, 5.0 x 108, 5.2 x 108, 5.4 x 108, 5.5 x 108, 5.6 x 108,
5.8 x 108, 5.9 x 108, or
6.0 x 108) viable CD19 specific CAR-T cells. In some embodiments, the dose is
a therapeutically
effective amount of viable CD19 specific CAR-T cells. In other embodiments,
the dose is a
clinically effective amount of viable CD19 specific CAR-T cells. In some
embodiments, the
CD19 specific CAR of the cells is the same CD19 specific CAR as
tisagenlecleucel
(KYMRIAETD), a structural equivalent thereof, or a functional equivalent
thereof.
1009441 In some embodiments, a single dose of any of the CD19 specific CAR-T
cells described
herein includes about 50 x 106 to about 110x 106 (e.g., 50x 106, Six 106, 52x
106,53 x 106,54
x 106, 55 x 106, 56 x 106, 57 x 106, 58 x 106, 59 x 106, 60 x 106, 61 x 106,
62 x 106, 63 x 106, 64 x
106, 65 x 106, 66 x 106, 67 x 106, 68 x 106, 69 x 106, 70 x 106, 71 x 106, 72
x 106, 73 x 106, 74 x
106, 75 x 106, 76 x 106, 77 x 106, 78 x 106, 79 x 106, 80 x 106, 81 x 106, 82
x 106, 83 x 106, 84 x
106, 85 x 106, 86 x 106, 87 x 106, 88 x 106, 89 x 106, 90 x 106, 91 x 106, 92
x 106, 93 x 106, 94 x
106, 95 x 106, 96 x 106, 97 x 106, 98 x 106, 99 x 106, 100 x 106, 101 x 106,
102 x 106, 103 x 106,
104x 106, 105x 106, 106x 106, 107x 106, 108x 106, 109x 106, or 110 x 106)
viable CD19
specific CAR-T cells. In some embodiments, the dose is a therapeutically
effective amount of
viable CD19 specific CAR-T cells. In other embodiments, the dose is a
clinically effective
amount of viable CD19 specific CAR-T cells. In some embodiments, the viable
CD19 specific
CAR-T cells include CD19 specific CAR expressing CD4+ T cells and CD19
specific CAR
expressing CD8+ T cells at a ratio of about 1:1. In some embodiments, the CD19
specific CAR
is the same CD19 specific CAR as lisocabtagene maraleucel (BREYANZP)), a
structural
equivalent thereof, or a functional equivalent thereof.
277
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009451 In some embodiments, a single dose of any of the CD19 specific CAR-T
cells described
herein includes about 2 x 108 viable CD19 specific CAR-T cells. In some
embodiments, a single
infusion bag of any of the CD19 specific CAR-T cells described herein includes
about 2 x 108
viable CD19 specific CAR-T cells in a cell suspension of about 68 mL. In some
embodiments,
the CD19 specific CAR is the same CD19 specific CAR as axicabtagene ciloleucel

(YESCARTA ), a structural equivalent thereof, or a functional equivalent
thereof.
1009461 In some embodiments, a single dose of any of the CD19 specific CAR-T
cells described
herein includes about 2 x 108 viable CD19 specific CAR-T cells. In some
embodiments, a single
infusion bag of any of the CD19 specific CAR-T cells described herein includes
about 2 x 108
viable CD19 specific CAR-T cells in a cell suspension of about 68 mL. In some
embodiments,
the CD19 specific CAR is the same CD19 specific CAR as brexucabtagene
autoleucel
(TECARTUS ), a structural equivalent thereof, or a functional equivalent
thereof
1009471 In some embodiments, a single dose of any of the CD19 specific CAR-T
cells described
herein includes about 0.2 x 106 to about 5.0 x 106 (e.g., about 0.2 x 106, 0.3
x 106, 0.4 x 106, 0.5 x
106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1.0 x 106, 1.1 x 106, 1.2 x
106, 1.3 x 106, 1.4 x 106,
1.5 x 106, 1.6x 106, 1.7 x 106, 1.8 x 106, 1.9x 106, 2.0x 106, 2.1 x 106,2.2 x
106, 2.3 x 106, 2.4 x
106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3.0 x 106, 3.1 x
106, 3.2 x 106, 3.3 x 106,
3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x 106, 3.9 x 106, 4.0 x 106,
4.1 x 106, 4.2 x 106, 4.3 x
106, 4.4 x 106, 4.5 x 106, 4.6 x 106, 4.7 x 106, 4.8 x 106, 4.9 x 106, or 5.0
x 106) viable CD19
specific CAR-T cells per kg of body weight for a subject with a body weight of
50 kg or less. In
some embodiments, a single dose of any of the CD19 specific CAR-T cells
described herein
includes about 0.1 x 108 to about 2.5 x 108 (e.g., about 0.1 x 106, 0.2 x 106,
0.3 x 106, 0.4 x 106,
0.5 x 106, 0.6 x 106, 0.7 x 106, 0.8 x 106, 0.9 x 106, 1.0 x 106, 1.1 x 106,
1.2 x 106, 1.3 x 106, 1.4 x
106, 1.5 x 106, 1.6 x 106, 1.7 x 106, 1.8 x 106, 1.9 x 106, 2.0 x 106, 2.1 x
106, 2.2 x 106, 2.3 x 106,
2.4 x 106, or 2.5 x 106) viable CD19 specific CAR-T cells per kg of body
weight for a subject
with a body weight of more than 50 kg. In some embodiments, a single dose of
any of the CD19
specific CAR-T cells described herein includes about 0.6 x 108 to about 6.0 x
108 (e.g., about 0.6
x 108, 0.7 x 108, 0.8 x 108, 0.9 x 108, 1.0 x 108, 1.1 x 108,1.2 x 108, 1.3 x
108, 1.4 x 108, 1.5 x 108,
1.6 x 108, 1.7 x 108, 1.8 x 108, 1.9 x 108, 2.0 x 108, 2.1 x 108, 2.2 x 108,
2.3 x 108, 2.4 x 108, 2.5 x
108, 2.6 x 108, 2.7 x 108, 2.8 x 108, 2.9 x 108, 3.0 x 108, 3.1 x 108, 3.2 x
108, 3.3 x 108, 3.4 x 108,
278
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
3.5 x 108, 3.6 x 108, 3.7 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.1 x 108,
4.2 x 108, 4.3 x 108, 4.4 x
108, 4.5 x 108, 4.6 x 108, 4.7 x 108, 4.8 x 108, 4.9 x 108, 5.0 x 108, 5.1 x
108, 5.2 x 108, 5.3 x 108,
5.4 x 108, 5.5 x 108, 5.6 x 108, 5.7 x 108, 5.8 x 108, 5.9 x 108, or 6.0 x
108) viable CD19 specific
CAR-T cells. In some embodiments, a single infusion bag of any of the CD19
specific CAR-T
cells described herein includes about 0.6 x 108 to about 6.0 x 108 (e.g.,
about 0.6 x 108, 0.7 x 108,
0.8 x 108, 0.9 x 108, 1.0 x 108, 1.1 x 108, 1.2 x 108, 1.3 x 108, 1.4 x 108,
1.5 x 108, 1.6 x 108, 1.7 x
108, 1.8 x 108, 1.9 x 108, 2.0 x 108, 2.1 x 108, 2.2 x 108, 2.3 x 108, 2.4 x
108, 2.5 x 108, 2.6 x 108,
2.7 x 108, 2.8 x 108, 2.9 x 108, 3.0 x 108, 3.1 x 108, 3.2 x 108, 3.3 x 108,
3.4 x 108, 3.5 x 108, 3.6 x
108, 3.7 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.1 x 108, 4.2 x 108, 4.3 x
108, 4.4 x 108, 4.5 x 108,
4.6 x 108, 4.7 x 108, 4.8 x 108, 4.9 x 108, 5.0 x 108, 5.1 x 108, 5.2 x 108,
5.3 x 108, 5.4 x 108, 5.5 x
108, 5.6 x 108, 5.7 x 108, 5.8 x 108, 5.9 x 108, or 6.0 x 108) viable CD19
specific CAR-T cells in a
cell suspension of from about 10 mL to about 50 mL. In some embodiments, the
dose is a
therapeutically effective amount of viable CD19 specific CAR-T cells. In other
embodiments,
the dose is a clinically effective amount of viable CD19 specific CAR-T cells.
In some
embodiments, the CD19 specific CAR of the cells is the same CD19 specific CAR
as
tisagenlecleucel (KYMRIAEC), a structural equivalent thereof, or a functional
equivalent
thereof.
1009481 In some embodiments, the BCMA specific (BCMA) CAR-T cells described
herein are
administered to a subject at a dose of about 250 x 106 to about 500 x 106
(e.g., 250 x 106, 255 x
106, 260 x 106, 265 x 106, 270 x 106, 275 x 106, 280 x 106, 285 x 106, 290 x
106, 295 x 106, 300 x
106, 305 x 106, 310 x 106, 315 x 106, 320 x 106, 325 x 106, 330 x 106, 335 x
106, 340 x 106, 345 x
106, 350 x 106, 355 x 106, 360 x 106, 365 x 106, 370 x 106, 375 x 106, 380 x
106, 385 x 106, 390 x
106, 395 x 106, 400 x 106, 405 x 106, 410 x 106, 415 x 106, 420 x 106, 425 x
106, 430 x 106, 435 x
106, 440 x 106, 445 x 106, 450 x 106, 455 x 106, 460 x 106, 465 x 106, 470 x
106, 475 x 106, 480 x
106, 485 x 106, 490 x 106, 495 x 106, or 500 x 106) viable BCMA specific CAR-T
cells. In some
embodiments, the dose is a therapeutically effective amount of viable BCMA
specific CAR-T
cells. In other embodiments, the dose is a clinically effective amount of
viable BCMA specific
CAR-T cells. In some embodiments, the viable BCMA specific CAR-T cells include
BCMA
specific CAR expressing CD4+ T cells and BCMA specific CAR expressing CD8+ T
cells at a
ratio of about 1:1. In some embodiments, the BCMA specific CAR of the cells is
idecabtagene
vicleucel (ABECEMA''), a structural equivalent thereof, or a functional
equivalent thereof.
279
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
1009491 In some embodiments, a subject is administered about 250 x 106 to
about 500 x 106
(e.g., 250 x 106, 255 x 106, 260 x 106, 265 x 106, 270 x 106, 275 x 106, 280 x
106, 285 x 106, 290
x 106, 295 x 106, 300 x 106, 305 x 106, 310 x 106, 315 x 106, 320 x 106, 325 x
106, 330 x 106, 335
x 106, 340 x 106, 345 x 106, 350 x 106, 355 x 106, 360 x 106, 365 x 106, 370 x
106, 375 x 106, 380
x 106, 385 x 106, 390 x 106, 395 x 106, 400 x 106, 405 x 106, 410 x 106, 415 x
106, 420 x 106, 425
x 106, 430 x 106, 435 x 106, 440 x 106, 445 x 106, 450 x 106, 455 x 106, 460 x
106, 465 x 106, 470
x 106, 475 x 106, 480 x 106, 485 x 106, 490 x 106, 495 x 106, or 500 x 106)
viable BCMA specific
CAR-T cells described herein. In some embodiments, the dose is a
therapeutically effective
amount of viable BCMA specific CAR-T cells. In other embodiments, the dose is
a clinically
effective amount of viable BCMA specific CAR-T cells. In some instances, 50%
of the viable
BCMA specific CAR-T cells are BCMA specific CAR expressing CD4+ T cells and
50% of the
viable BCMA specific CAR-T cells are BCMA specific CAR expressing CD8+ T
cells. In some
embodiments, the BCMA specific CAR of the cells is idecabtagene vicleucel
(ABECEMA ), a
structural equivalent thereof, or a functional equivalent thereof.
1009501 In some embodiments, the BCMA specific CAR-T cells described herein
are
administered to a subject at a dose of up to about 5 x 108 viable BCMA
specific CAR-T cells. In
some embodiments, a subject is administered from about 2.5 x 108 to about 5.0
x 108 (e.g., about
0.2 x 108, 0.4 x 108, 0.5 x 108, 0.6 x 108, 0.8 x 108, 0.9 x 108, 1.0 x 108,
1.2 x 108, 1.4 x 108, 1.5 x
108, 1.6 x 108, 1.8 x 108, 1.9 x 108, 2.0 x 108, 2.2 x 108, 2.4 x 108, 2.5 x
108, 2.6 x 108, 2.8 x 108,
2.9 x 108, 3.0 x 108, 3.2 x 108, 3.4 x 108, 3.5 x 108, 3.6 x 108, 3.8 x 108,
3.9 x 108, 4.0 x 108, 4.2 x
108, 4.4 x 108, 4.5 x 108, 4.6 x 108, 4.8 x 108, 4.9 x 108, or 5.0 x 108)
viable BCMA specific
CAR-T cells per kg of body weight. In some embodiments, the dose is a
therapeutically
effective amount of viable BCMA specific CAR-T cells. In other embodiments,
the dose is a
clinically effective amount of viable BCMA specific CAR-T cells. In some
embodiments, the
BCMA specific CAR of the cells is the same BCMA specific CAR as idecabtagene
vicleucel
(ABECEMAP), a structural equivalent thereof, or a functional equivalent
thereof
1009511 In some embodiments, a single dose of any of the BCMA specific CAR-T
cells
described herein includes about 250 x 106 to about 500 x 106 (e.g., 250 x 106,
255 x 106, 260 x
106, 265 x 106, 270 x 106, 275 x 106, 280 x 106, 285 x 106, 290 x 106, 295 x
106, 300 x 106, 305 x
106, 310 x 106, 315 x 106, 320 x 106, 325 x 106, 330 x 106, 335 x 106, 340 x
106, 345 x 106, 350 x
280
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
106, 355 x 106, 360 x 106, 365 x 106, 370 x 106, 375 x 106, 380 x 106, 385 x
106, 390 x 106, 395 x
106, 400 x 106, 405 x 106, 410 x 106, 415 x 106, 420 x 106, 425 x 106, 430 x
106, 435 x 106, 440 x
106, 445 x 106, 450 x 106, 455 x 106, 460 x 106, 465 x 106, 470 x 106, 475 x
106, 480 x 106, 485 x
106, 490 x 106, 495 x 106, or 500 x 106) viable BCMA specific CAR-T cells. In
some
embodiments, the dose is a therapeutically effective amount of viable BCMA
specific CAR-T
cells. In other embodiments, the dose is a clinically effective amount of
viable BCMA specific
CAR-T cells. In some embodiments, the viable BCMA specific CAR-T cells include
BCMA
specific CAR expressing CD4+ T cells and BCMA specific CAR expressing CD8+ T
cells at a
ratio of about 1:1. In some embodiments, the BCMA specific CAR is the same
BCMA specific
CAR as idecabtagene vicleucel (ABECEMA4), a structural equivalent thereof, or
a functional
equivalent thereof
1009521 In some embodiments, a single dose of any of the BCMA specific CAR-T
cells
described herein includes about 250 x 106 to about 500 x 106 (e.g., 250 x 106,
255 x 106, 260 x
106, 265 x 106, 270 x 106, 275 x 106, 280 x 106, 285 x 106, 290 x 106, 295 x
106, 300 x 106, 305 x
106, 310 x 106, 315 x 106, 320 x 106, 325 x 106, 330 x 106, 335 x 106, 340 x
106, 345 x 106, 350 x
106, 355 x 106, 360 x 106, 365 x 106, 370 x 106, 375 x 106, 380 x 106, 385 x
106, 390 x 106, 395 x
106, 400 x 106, 405 x 106, 410 x 106, 415 x 106, 420 x 106, 425 x 106, 430 x
106, 435 x 106, 440 x
106, 445 x 106, 450 x 106, 455 x 106, 460 x 106, 465 x 106, 470 x 106, 475 x
106, 480 x 106, 485 x
106, 490 x 106, 495 x 106, or 500 x 106) viable BCMA specific CAR-T cells per
kg of body
weight. In some embodiments, a single dose of any of the BCMA specific CAR-T
cells
described herein includes about 2.5 x 108 to about 5.0 x 108 (e.g., about 0.2
x 108, 0.4 x 108, 0.5 x
108, 0.6 x 108, 0.8 x 108, 0.9 x 108, 1.0 x 108, 1.2 x 108, 1.4 x 108, 1.5 x
108, 1.6 x 108, 1.8 x 108,
1.9 x 108, 2.0 x 108, 2.2 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.8 x 108,
2.9 x 108, 3.0 x 108, 3.2 x
108, 3.4 x 108, 3.5 x 108, 3.6 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.2 x
108, 4.4 x 108, 4.5 x 108,
4.6 x 108, 4.8 x 108, 4.9 x 108, or 5.0 x 108) viable BCMA specific CAR-T
cells per kg of body
weight. In some embodiments, a single dose of any of the BCMA specific CAR-T
cells
described herein includes about 2.5 x 108 to about 5.0 x 108 (e.g., about 0.2
x 108, 0.4 x 108, 0.5 x
108, 0.6 x 108, 0.8 x 108, 0.9 x 108, 1.0 x 108, 1.2 x 108, 1.4 x 108, 1.5 x
108, 1.6 x 108, 1.8 x 108,
1.9 x 108, 2.0 x 108, 2.2 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.8 x 108,
2.9 x 108, 3.0 x 108, 3.2 x
108, 3.4 x 108, 3.5 x 108, 3.6 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.2 x
108, 4.4 x 108, 4.5 x 108,
4.6 x 108, 4.8 x 108, 4.9 x 108, or 5.0 x 108) viable BCMA specific CAR-T
cells. In some
281
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
embodiments, a single infusion bag of any of the BCMA specific CAR-T cells
described herein
includes about 2.5 x 108 to about 5.0 x 108 (e.g., about 0.2 x 108, 0.4 x 108,
0.5 x 108, 0.6 x 108,
0.8 x 108, 0.9 x 108, 1.0 x 108, 1.2 x 108, 1.4 x 108, 1.5 x 108, 1.6 x 108,
1.8 x 108, 1.9 x 108, 2.0 x
108, 2.2 x 108, 2.4 x 108, 2.5 x 108, 2.6 x 108, 2.8 x 108, 2.9 x 108, 3.0 x
108, 3.2 x 108,34 x 108,
3.5 x 108, 3.6 x 108, 3.8 x 108, 3.9 x 108, 4.0 x 108, 4.2 x 108, 4.4 x 108,
4.5 x 108, 4.6 x 108, 4.8 x
108, 4.9 x 108, or 5.0 x 108) viable BCMA specific CAR-T cells in a cell
suspension of from
about 10 mL to about 500 mL. In some embodiments, the cell suspension is about
50 mL, 250
mL, or about 500 mL. In some embodiments, the dose is a therapeutically
effective amount of
viable BCMA specific CAR-T cells. In other embodiments, the dose is a
clinically effective
amount of viable BCMA specific CAR-T cells. In some embodiments, the BCMA
specific CAR
of the cells is the same BCMA specific CAR as idecabtagene vicleucel
(ABECEMAP), a
structural equivalent thereof, or a functional equivalent thereof.
BB. Methods for Administering Hypoimmunogenic Cells Including
T Cells
1009531 As is described in further detail herein, provided herein are methods
for treating a
patient with a condition, disorder, or disorder through administration of
hypoimmunogenic cells,
particularly hypoimmunogenic T cells. As will be appreciated, for all the
multiple embodiments
described herein related to the timing and/or combinations of therapies, the
administration of the
cells is accomplished by a method or route which results in at least partial
localization of the
introduced cells at a desired site. The cells can be infused, implanted, or
transplanted directly to
the desired site, or alternatively be administered by any appropriate route
which results in
delivery to a desired location in the subject where at least a portion of the
implanted cells or
components of the cells remain viable.
1009541 Provided herein are methods for treating a patient with a condition,
disorder, or disorder
includes administration of a population of hypoimmunogenic cells (e.g.,
primary T cells, T cells
differentiated from hypoimmunogenic induced pluripotent stem cells, or other
cells differentiated
from hypoimmunogenic induced pluripotent stem cells described herein) to a
subject, e.g., a
human patient. For instance, a population of hypoimmunogenic primary T cells
such as, but
limited to, CD3+ T cells, CD4+ T cells, CD8+ T cells, naïve T cells,
regulatory T (Treg) cells,
non-regulatory T cells, Thl cells, Th2 cells, Th9 cells, Th17 cells, T-
follicular helper (Tfh) cells,
cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm)
cells, effector
282
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
memory T (Tern) cells, effector memory T cells that express CD45RA (TEMRA
cells), tissue-
resident memory (Trm) cells, virtual memory T cells, innate memory T cells,
memory stem cell
(Tsc), -y6 T cells, and any other subtype of T cell is administered to a
patient to treat a condition,
disorder, or disorder. In some embodiments, an immunosuppressive and/or
immunomodulatory
agent (such as, but not limited to a lymphodepletion agent) is not
administered to the patient
before the administration of the population of hypoimmunogenic cells. In some
embodiments, an
immunosuppressive and/or immunomodulatory agent is administered at least 1, 2,
3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14 days or more before the administration of the cells. In
some embodiments,
an immunosuppressive and/or immunomodulatory agent is administered at least 1
week, 2
weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks
or more
before the administration of the cells. In numerous embodiments, an
immunosuppressive and/or
immunomodulatory agent is not administered to the patient after the
administration of the cells,
or is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days
or more after the
administration of the cells. In some embodiments, an immunosuppressive and/or
immunomodulatory agent is administered at least 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6
weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more after the administration of
the cells. In
some embodiments where an immunosuppressive and/or immunomodulatory agent is
administered to the patient before or after the administration of the cells,
the administration is at
a lower dosage than would be required for cells with one or more MEC I and/or
MHC II
molecule expression and without exogenous expression of CD47.
1009551 Non-limiting examples of an immunosuppressive and/or immunomodulatory
agent
(such as, but not limited to a lymphodepletion agent) include cyclosporine,
azathioprine,
mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone,
methotrexate,
gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,
15-deoxyspergualine,
6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-
thymocyte
globulin, thymopentin, thymosin-a and similar agents. In some embodiments, the

immunosuppressive and/or immunomodulatory agent is selected from a group of
immunosuppressive antibodies consisting of antibodies binding to p75 of the IL-
2 receptor,
antibodies binding to, for instance, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40,
CD45, IFN-
gamma, TNF-alpha, IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, 1L-7, IL-8, IL-10, CD11
a, or CD58,
and antibodies binding to any of their ligands. In some embodiments, such an
283
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
immunosuppressive and/or immunomodulatory agent may be selected from soluble
IL-15R, IL-
10, B7 molecules (e.g., B7-1, B7-2, variants thereof, and fragments thereof),
ICOS, and 0X40,
an inhibitor of a negative T cell regulator (such as an antibody against CTLA-
4) and similar
agents.
1009561 In some embodiments, where an immunosuppressive and/or
immunomodulatory agent
is administered to the patient before or after the administration of the
cells, the administration is
at a lower dosage than would be required for cells with one or mole MEC I
and/or MEC II
molecule expression, TCR expression and without exogenous expression of CD47.
In some
embodiments, where an immunosuppressive and/or immunomodulatory agent is
administered to
the patient before or after the first administration of the cells, the
administration is at a lower
dosage than would be required for cells with one or more MEC I and/or MHC II
molecule
expression, TCR expression and without exogenous expression of CD47.
1009571 In some embodiments, the cells described are co-administered with a
therapeutic agent
that that binds to and/or interacts with one or more receptors selected from
the group consisting
of CD94, KIR2DL4, PD-1, an inhibitory NK cell receptor, and an activating NK
receptor. In
some instances, the therapeutic agent binds to a receptor on the surface of an
NK cell, including
one or more subpopulations of NK cells. In some embodiments, the therapeutic
agent is selected
from the group consisting of an antibody and fragments and variants thereof,
an antibody
mimetic, a small molecule, a blocking peptide, and a receptor antagonist.
1009581 For therapeutic application, cells prepared according to the disclosed
methods can
typically be supplied in the form of a pharmaceutical composition comprising
an isotonic
excipient, and are prepared under conditions that are sufficiently sterile for
human
administration. For general principles in medicinal formulation of cell
compositions, see "Cell
Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy,"
by Morstyn
& Sheridan eds, Cambridge University Press, 1996; and "Hematopoietic Stem Cell
Therapy," E.
D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The cells can be
packaged in a device
or container suitable for distribution or clinical use.
1009591 In some embodiments, the cells described herein are contraindicated in
patients with
known Type I hypersensitivity or anaphylactic reactions to murine proteins,
Chinese Hamster
Ovary (CHO) cell proteins, or to any component of the compositions described
herein. In some
embodiments, the cells described herein are contraindicated in patients who
have or have had
284
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
progressive multifocal leukoencephalopathy (PML). In some embodiments, the
cells described
herein are not recommended for use in patients with severe, active infections.
1009601 In some embodiments, the cells described herein are administered to a
subject with an
autoimmune disease/disorder and/or inflammatory disease/disorder who has been
previously
treated with rituximab (RITUXANg). In some embodiments, the cells described
herein are
administered to a subject with an autoimmune disease/disorder and/or
inflammatory
disease/disorder who has been previously treated with rituximab (RITUXANO) and
has failed
and/or not responded to the rituximab treatment. In some embodiments, the
patent has
rheumatoid arthritis (RA). In some embodiments, the patient has RA and the
rituximab
treatment is in combination with methotrexate. In some embodiments, the
patient is an adult
patient that has moderately-to severely-active RA. In some embodiments, the
patient is an adult
patient that has moderately-to severely-active RA and the rituximab treatment
is in combination
with methotrexate. In some embodiments, the patient is an adult patient that
has moderately-to
severely-active RA who has inadequate response to one or more TNF antagonist
therapies and
the rituximab treatment is in combination with methotrexate. In some
embodiments, the
rituximab dose for RA in combination with methotrexate is two-1000 mg
intravenous infusions
separated by 2 weeks (one course) every 24 weeks and/or based on clinical
evaluation, but not
sooner than every 16 weeks. In some embodiments, the Methylprednisolone 100 mg
intravenous
or equivalent glucocorticoid is recommended 30 minutes prior to each infusion.
1009611 In some embodiments, the cells described herein are administered to a
subject with an
autoimmune disease/disorder and/or inflammatory disease/disorder who has been
previously
treated with rituximab (RITUXANO). In some embodiments, the cells described
herein are
administered to a subject with an autoimmune disease/disorder and/or
inflammatory
disease/disorder who has been previously treated with rituximab (RITUXANe) and
has failed
and/or not responded to the rituximab treatment. In some embodiments, the
patent has
granulomatosis with polyangiitis (GPA) (Wegener's Granulomatosis). In some
embodiments, the
patent has Microscopic polyangiitis (MPA) in adult patients in combination
with glucocorticoids.
In some embodiments, the rituximab dose for GPA and MPA in combination with
glucocorticoids is 375 mg/m2 once weekly for 4 weeks. In some embodiments, the
rituximab is
285
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
administered as a 100 mg/10 mL solution in a single-use vial. In some
embodiments, the
rituximab is administered as a 500 mg/50 mL solution in a single-use vial.
CC. Methods for Detecting the Presence of Antibodies
1009621 In some embodiments, biological samples from a patient are assayed to
determine
whether the samples comprise antibodies against one or more Y chromosome
genes.
1009631 Methods of detecting antibodies in a biological sample are well known
in the art. In
some embodiments, anti-Protocadherin-11 Y-linked antibodies and/or anti-
Neuroligin-4 Y-
linked are detected from a biological sample obtained from a patient using any
well- established
method in the art, such as enzyme-linked immunosorbent assay (ELISA), nucleic
acid and
testing, western blotting, and the like.
1009641 In some embodiments, the biological sample is a plasma sample, a
peripheral blood
sample, a urine sample, a sputum/saliva sample, or a serum sample obtained
from the patient.
1009651 In some embodiments, the methods are used to determine the appropriate
cell-based
therapy to administer to a patient with a disease or condition who would
benefit from a cell-
based therapy. In some embodiments, the methods are used to identify a patient
with a disease or
condition who would benefit from a cell-based therapy comprising reduced
expression of one or
more Y chromosome genes. In some embodiments, the methods are used to
determine whether a
cell-based therapy that does not comprise reduced expression of Protocadherin-
11 Y-linked
and/or of Neuroligin-4 Y-linked is susceptible to NK mediated cytotoxicity
upon administration
to a patient. In some embodiments, the methods are used to determine whether a
cell-based
therapy that does not comprise reduced expression of Protocadherin-11 Y-linked
and/or of
Neuroligin-4 Y-linked is susceptible to lysis by mature NK cells upon
administration to a patient.
In some embodiments, the methods are used to determine whether a cell-based
therapy that does
not comprise reduced expression of Protocadherin-11 Y-linked and/or of
Neuroligin-4 Y-linked
is susceptible to macrophage engulfment upon administration to a patient. In
some embodiments,
the methods are used to determine whether a cell-based therapy that does not
comprise reduced
expression of Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-linked is
susceptible to an
induced immune response upon administration to a patient. In some embodiments,
the methods
are used to determine whether a cell-based therapy that does not comprise
reduced expression of
Protocadherin-11 Y-linked and/or of Neuroligin-4 Y-linked is susceptible to an
induced
286
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
antibody-based immune response upon administration to a patient. In some
embodiments, the
methods are used in a method of treating a patient with a disease or condition
who would benefit
from a cell-based therapy.
[00966] In some embodiments, the methods comprise: (a) determining whether a
biological
sample from the patient comprises antibodies against one or more Y chromosome
genes by: (i)
obtaining or having obtained a biological sample from the patient; (ii)
performing or having
peifoimed an assay to determine whether antibodies against Piotocadheiin-11 Y-
linked are
present in the biological sample; and (iii) performing or having performed an
assay to determine
whether antibodies against Neuroligin-4 Y-linked are present in the biological
sample; and (b)
administering a population of the engineered cells, hypoimmunogenic T cells,
non-activated T
cells, pancreatic islet cells, cardiac muscle cells, or glial progenitor cells
of any one of claims 1-
50 to the patient, wherein: (i) if antibodies against Protocadherin-11 Y-
linked are present in the
biological sample, the population of cells comprises reduced expression of
Protocadherin-11 Y-
linked; (ii) if antibodies against Neuroligin-4 Y-linked are present in the
biological sample, the
population of cells comprises reduced expression of Neuroligin-4 Y-linked;
(iii) if antibodies
against Protocadherin-11 Y-linked and antibodies against Neuroligin-4 Y-linked
arc present in
the biological sample, the population of cells comprises reduced expression of
Protocadherin-11
Y-linked and of Neuroligin-4 Y-linked; (iv) if neither antibodies against
Protocadherin-11 Y-
linked nor antibodies against Neuroligin-4 Y-linked are present in the
biological sample, the
population of cells does not comprise reduced expression of Protocadherin-11 Y-
linked or of
Neuroligin-4 Y-linked.
IV. EXAMPLES
Example 1: Expression of Protocadherin-Y and Neuroligin-Y on iPSCs and on T
cells
iPSCs
[00967] To determine whether Protocadherin-Y and/or Neuroligin-Y were
expressed on iPSCs,
iPSCs derived from rhesus males were analyzed for Protocadherin-Y and
Neuroligin-Y
expression using standard techniques. The iPSCs were analyzed by flow
cytometry (using
standard methods). iPSCs derived from human females served as controls.
[00968] Cells were blocked with anti-Fc receptor antibodies and stained with
an anti-
Protocadherin-Y antibody or an anti-Neuroligin-Y antibody that was
concentration matched to
287
CA 03225283 2024- 1- 8

WO 2023/287827
PCT/US2022/036874
an isotype control. As shown in FIGs. lA and 1B, both Protocadherin-Y and
Neuroligin-Y were
expressed on iPSCs derived from male donors. Neither Protocadherin-Y nor
Neuroligin-Y were
expressed on iPSCs derived from female donors.
1009691 Without being bound by theory, the surprising finding that
Protocadherin-Y and
Neuroligin-Y are expressed on iPSCs suggests that Y chromosome-encoded
antigens could be
recognized in female recipients as foreign antigens, leading to T and B cell
activation.
T cells
1009701 To determine whether Protocadherin-Y and/or Neuroligin-Y were
expressed on T cells,
T cells from 5 male donors were sorted for CD3 expression to generate a CD3+
population, and
the CD3+ T cells were analyzed for Protocadherin-Y and Neuroligin-Y expression
using
standard techniques. The T cells were analyzed by flow cytometry (using
standard methods) after
thawing. CD3+ T cells from two female donors served as controls.
1009711 Cells were blocked with anti-Fc receptor antibodies and stained with
an anti-
Protocadherin-Y antibody or an anti-Neuroligin-Y antibody that was
concentration matched to
an isotype control. As shown in FIGs. 2A, 2B, and 2C, both Protocadherin-Y and
Neuroligin-Y
were expressed on T cells derived from male donors. Neither Protocadherin-Y
nor Neuroligin-Y
were expressed on T cells derived from female donors.
1009721 Without being bound by theory, the surprising finding that
Protocadherin-Y and
Neuroligin-Y are expressed on T cells suggests that Y chromosome-encoded
antigens could be
recognized in female recipients as foreign antigens, leading to T and B cell
activation.
ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent
cytotoxicity)
1009731 Male HIP T cells (=HLA-I/II KO; CD47 KI) were incubated with serum
from males,
females, females after pregnancies with girls, and females after pregnancies
with boys. Killing of
HIP T cells by CDC and ADCC mechanism was analyzed using Xcelligence cell
killing assay.
1009741 As shown in Figures 3-5, male-derived HIP T cells were killed by NK
cells and by
CDC in serum from females that had anti-H-Y antibodies, i.e. females that were
sensitized by
previous boy but not girl pregnancies.
288
CA 03225283 2024- 1- 8